James debuted in WWE on the October 10, 2005 episode of Raw as a face under the gimmick of WWE Women's Champion Trish Stratus' biggest fan. The angle had the two Divas competing together in tag team matches, with James' character becoming increasingly obsessed with Stratus. The storyline included a Halloween costume contest, in which James was dressed like Stratus and helped Stratus retain the Women's Championship in a Fulfill Your Fantasy battle royal at Taboo Tuesday by eliminating herself and Victoria at the same time. James even began utilizing Stratus' signature finishing moves as her own during matches. James later became the number one contender for the WWE Women's Championship on December 12, by defeating Victoria in a match to determine who would face Stratus at New Year's Revolution. In the championship match at the pay-per-view, James lost to Stratus.
Despite the defeat, James continued to be enamored of Stratus, which made Stratus uncomfortable. On March 6, 2006, the storyline had Stratus confronting James, telling her that they needed time apart from each other. Through the early part of 2006, Mickie would attack Ashley Massaro several times due to Massaro calling her "crazy". At the Royal Rumble pay-per-view, James defeated Massaro with then-Women's Champion, Trish Stratus, as the special guest referee. James would also confess her love for Stratus at the event. Massaro got revenge one week later on Raw.
James and Stratus teamed together at the March 18, 2006 episode of Saturday Night's Main Event to defeat Candice Michelle and Victoria. After the match, James attacked Stratus and vowed to destroy her, becoming a villain in the process for the first time in her WWE career. The feud between James and Stratus culminated in a Women's Championship match at WrestleMania 22, which James won, to earn her first Women's Championship in what was looked at as one of the best female matches ever produced at WrestleMania. Her angle with Stratus continued into Backlash during a rematch, after Stratus legitimately dislocated her shoulder when James threw her out of the ring. The feud would come to an end on the June 26 episode of Raw, when Mickie defeated Stratus in a Women's Championship match.
James dropped the WWE Women's Championship on August 14 to Lita, after Lita hit James with the title belt. After the Women's Championship was vacated due to the retirement of Trish Stratus, James entered a tournament to determine the new champion. She defeated Victoria and Melina en route to the finals at Cyber Sunday, where she lost to Lita. James transitioned into a face after she and Lita wrestled in a series of matches in which Lita chose stipulations to hinder Mickie's wrestling ability. The feud between James and Lita ended at Survivor Series, where James defeated Lita, in the latter's retirement match, to win her second Women's Championship.
James then began an angle with Melina on January 29, 2007, when Melina became the number one contender for the Women's Championship. Following a successful title defense on February 5, James teamed with Super Crazy in a mixed tag team match against Melina and Johnny Nitro. After Melina pinned James for the victory, she challenged her to a rematch for the title. James would subsequently lose the Women's Championship to Melina on February 19 and, in continuation of their storyline feud, failed to regain the title during the first women's Falls Count Anywhere match in WWE history. During the finish of the match, James fell from the top turnbuckle and landed on her neck, which resulted in a rushed finish. James, however, was not seriously injured in the incident.
The scripted feud between James and Melina was rekindled during her photo op on Raw. At a house show in Paris, France on April 24, James won her third Women's Championship during a triple threat match that also involved Victoria. Since Mickie pinned Victoria and not Melina, an immediate rematch was scheduled, in which James dropped the title back to Melina, giving her the shortest Women's Championship reign in WWE history. James later received a rematch for the title at Backlash, but was unsuccessful. After Backlash, James would only make sporadic appearances on television, wrestling occasionally in tag-matches and rarely in singles competition. On the November 26 episode of Raw, James defeated Melina in a number one contender's match for Beth Phoenix's Women's Championship, setting up a title match between the two at Armageddon, in which Phoenix retained the title.
On the April 14, 2008 episode of Raw, held in London, England, James defeated Beth Phoenix to win her fourth Women's Championship. At Judgment Day, James successfully defended her title against Melina and Beth Phoenix in a triple-threat match. Mickie re-entered the feud against Phoenix in mid-2008, where she and Kofi Kingston teamed up against Phoenix and Santino Marella at SummerSlam in a Winner Takes All tag team match for both the Women's and Intercontinental Championships, in which Mickie and Kingston lost their titles to Phoenix and Marella. After James lost the championship, she had two rematches for the title, but was unsuccessful in regaining it. At Survivor Series in November, James was part of the victorious Raw Diva team which defeated the SmackDown Divas in a five-on-five elimination match; she eliminated Michelle McCool, before being eliminated by Maryse. The following month at Armageddon, James teamed with Maria, Michelle McCool and Kelly Kelly in a winning effort against Jillian Hall, Maryse, Victoria and Natalya.
Following an appearance in the 25-Diva battle royal at WrestleMania XXV, James began feuding with the WWE Divas Champion Maryse heading into Night of Champions on July 26. At the event, James defeated Maryse to win her first Divas Championship, becoming only the second Diva in history to have held both the Women's and Divas titles. Throughout the Summer, Mickie successfully defended the title against Gail Kim and Beth Phoenix on episodes of Raw, and against Alicia Fox at the Hell in a Cell pay-per-view on October 4. Two weeks later on Raw, James lost the title to Jillian Hall after an approximate three month title reign. After the show, James was traded to the SmackDown brand for the first time in her career, due to a Diva trade made by Raw guest host Nancy O'Dell.
James made her debut with the brand on the October 23 episode of SmackDown, defeating Layla. On the October 30 episode of SmackDown, a controversial angle began that saw WWE Women's Champion Michelle McCool and Layla, collectively known as LayCool, bully James. On the November 20 episode of SmackDown, after James defeated Layla, McCool gave James the nickname "Piggy James", that sent James to tears, resulting in a five-on-five Survivor Series match at the November pay-per-view Survivor Series, where James' team prevailed over McCool's team, with James and Melina as the sole survivors. On the December 4 episode of SmackDown, James became the number one contender for McCool's Women's Championship by defeating Beth Phoenix and Natalya in a triple threat match. The following week, James challenged McCool for the championship at TLC: Tables, Ladders & Chairs, but was unsuccessful after interference from Layla. The storyline feud continued into the Royal Rumble on January 31, 2010 where James finally defeated McCool, in 20 seconds, to become a five-time Women's Champion, the second most reign with the old WWE Women's Championship, after Trish Stratus. Over the following couple of weeks, SmackDown consultant Vickie Guerrero was introduced into the rivalry, choosing to side with LayCool over James. On the February 26 episode of SmackDown, McCool used her rematch clause to face James for the Women's Championship, with Guerrero acting as special guest referee. After Guerrero slapped James, McCool pinned her to regain the title.
On March, James was diagnosed with a staph infection on her right knee, putting her out of action for three weeks. She returned on the March 22 episode of Raw, where alongside Kelly Kelly, she accompanied Eve Torres, Beth Phoenix, and Gail Kim in their losing effort against McCool, Maryse and Layla, who had Vickie Guerrero and Alicia Fox in their corner. This set up a 10-Diva tag team match at WrestleMania XXVI, in which James made an unsuccessful in-ring return after Vickie pinned Kelly. James made her last appearance on Raw during a rematch, where her team was victorious. James' final match in WWE aired on the April 23 episode of SmackDown where she teamed with former long-time rival Beth Phoenix against LayCool in a losing effort when she was pinned by Layla. James was released from WWE one day earlier on April 22, having pre-taped her SmackDown match.
In November 2013, James served as guest trainer at the WWE Performance Center in Orlando, Florida for a week, training NXT's female wrestlers. James also attended that week's NXT live event in Tampa, Florida.
On October 13, 2016, it was announced that James would return to WWE's developmental brand NXT at the NXT TakeOver: Toronto event on November 19, competing against Asuka for the NXT Women's Championship. On the October 26 episode of NXT, James cut a promo where she formally re–introduced herself and challenged Asuka for the championship. At the event, James was defeated and after the match Asuka refused to shake her hand as a sign of disrespect.
On December 8, it was announced that James had signed a multi–year contract with WWE. On the January 17, 2017 episode of SmackDown Live, James made her return by helping Alexa Bliss retain her SmackDown Women's Championship against Becky Lynch in a Steel Cage match, revealing herself as the woman under the "La Luchadora" gimmick and establishing herself as a villainess, while also allying with Bliss in the process. She competed in her first match in nearly seven years in a WWE ring on January 29, 2017 at the Royal Rumble pay–per–view, teaming with Bliss and Natalya against Lynch, Naomi, and Nikki Bella in a losing effort. James went on to face Lynch at the Elimination Chamber pay–per–view in another losing effort. She would defeat Lynch in a rematch on the edition of February 14 of SmackDown, but would lose a Two-Out-of-Three Falls match two weeks later to end the storyline. In March, James turned face after hitting Bliss with a Mick Kick, ending their brief alliance and leading to a non-title match between the two on the edition of March 14 of SmackDown, in which James was victorious. At WrestleMania 33, James took part in a six–pack challenge for Bliss’ SmackDown Women's Championship, which saw Naomi winning the title.
Shortly after WrestleMania, James was drafted to Raw as part of the 2017 Superstar Shake–Up. In October, after being named the No. 1 Contender by Raw General Manager Kurt Angle, James faced Bliss for the Raw Women's Championship at TLC: Tables, Ladders & Chairs, but was unsuccessful in capturing the title. A few weeks later on the October 30 edition of Raw, James once again challenged Bliss for the title in the main event but came up short. On January 28, 2018 at the Royal Rumble, James entered the first-ever women's Royal Rumble match at No. 26 and lasted over 8 minutes before being eliminated by long–time rival Trish Stratus. The following month, James competed in the first-ever women's Elimination Chamber match at the namesake pay–per–view, where she entered at No. 5 and eliminated Sonya Deville after a diving Lou Thesz press off the top of a pod before being eliminated by Bayley.
James proceeded to turn heel and realigned herself with Bliss on the February 26 Raw. At WrestleMania 34, James participated in the first-ever WrestleMania Women's Battle Royal for the WrestleMania Women’s Battle Royal Trophy, but was eliminated from the match by Ruby Riott. Later that night, she had planned to manage Bliss to her title defense against Nia Jax, but was attacked and ejected by Jax before the match began. In May, James competed unsuccessfully in a Triple Threat match against Bliss and Bayley, as well as a Gauntlet match in attempts to qualify for the women's Money in the Bank ladder match.
In August, James returned from a brief hiatus at the sides of Alexa Bliss and Alicia Fox. With Bliss, the tandem began a scripted feud with her former rivals and WWE Hall of Famers Trish Stratus and Lita, setting up a tag team match at WWE Evolution, WWE's first-ever all-women's pay–per–view. Just three days before the event on October 25, however, it was announced that Bliss was pulled out of the match due to a concussion, and that Fox would be her replacement. In the event's opening contest, James and Fox were unsuccessful against Stratus and Lita.
On November 18 at Survivor Series, James was selected by Bliss to be a part of Team Raw along with Jax, Tamina, Bayley and Sasha Banks to go against Team SmackDown’s Naomi, Carmella, Deville, Asuka and Mandy Rose. Team Raw came out victorious after Jax was the sole survivor. The victory also upped James’ undefeated streak to 5-0 at Survivor Series. The next night on Raw, James was selected by “General Manager-Elect” Baron Corbin to answer an open-challenge by Raw Women's Champion Ronda Rousey, but lost the title match via submission. Meanwhile, James and Bobby Lashley were announced as one of the teams that would be competing in season 2 of Mixed Match Challenge. Despite pulling ahead with a 3-0 lead on the competition, the duo lost to Bayley and Finn Bálor in the playoffs and were eliminated from the tournament.
Back on Raw, James made history on the edition of December 17 by competing in the most matches by a female superstar in the show's 25-year history with 164 total combined matches by this point. On January 27, 2019 at the Royal Rumble, James entered the second women's Royal Rumble match at number five and lasted over 11 minutes before being eliminated by Tamina. The following night on Raw, James once again aligned herself with Bliss in hopes of qualifying for the Elimination Chamber event to crown the first-ever WWE Women's Tag Team Champions, but the long-time allies lost their tag team match to Jax and Tamina.
James would later compete in the second-annual WrestleMania Women’s Battle Royal at WrestleMania 35 for the trophy, eliminating Mandy Rose from the match before subsequently being eliminated by Sonya Deville. On April 16 during the 2019 WWE Superstar Shake-up, James was drafted back to the SmackDown brand. During a live event on June 1, it was reported that James had suffered a torn ACL in a singles match with Carmella. On July 16, James underwent successful knee surgery and would be out of action for 7 to 9 months, which would be the remainder of her multi-year contract.
Beginning on the September 27, 2019 broadcast of WWE Main Event, James began commentating for the program alongside Dio Maddin and Vic Joseph, and later Maddin and Byron Saxton.
After fourteen months of injury, James returned on the August 10, 2020 episode of Raw as a face, only to be interrupted by Natalya and Lana. James then lost a singles match to Natalya by countout thanks to interference from Lana. On the August 31 episode of Raw, James confronted the Raw Women's Champion, Asuka, letting Asuka know that she was going after her title. During this exchange, Lana and Natalya interrupted, which led to a match between James and Lana, which James won. On the September 14 episode of Raw, James challenged Asuka for the Raw Women's Championship, which she lost after a botched finish due to the referee believing that James could not continue the match.
During the 2020 WWE Draft in October, James went undrafted. Her first post-draft appearance occurred on January 4, 2021, during the special "Legends Night" episode of Raw, appearing alongside several other WWE legends. That same month, James competed in the women’s Royal Rumble match entering at number nineteen, but was eliminated by Lacey Evans.
On April 15, 2021, WWE announced that they had released Mickie James along with several other talents.
On the January 7, 2022 episode of SmackDown, James was announced as a participant in the women's Royal Rumble match at the Royal Rumble. During the Royal Rumble, she entered at number twenty, where she eliminated her former rival Michelle McCool before being eliminated by Lita.