Barnett's documented professional debut was in early 1997 in Washington, competing for United Full Contact Federation. He won via rear-naked choke submission under three minutes into the fight. He would continue to dominate, reaching a 9-0 record with seven first-round stoppages and wins over Future UFC Hall of Famer Dan Severn, Bobby Hoffman, John Marsh, and Bob Gilstrap twice. With a 9-0 record, Barnett was invited to compete in the UFC.

Barnett made his UFC debut at UFC 28 on November 17, 2000 against 6' 10" Gan "The Giant" McGee and Barnett won via TKO in the second round. Despite suffering a KO loss in his next fight against Pedro Rizzo he would bounce back and win his next two fights over 7' 0" Dutch Kickboxer Semmy Schilt via armbar submission, and then received a submission win in a rematch with Bobby Hoffman. Subsequent to his win over Hoffman, Barnett tested positive for banned substances and was given a warning by the Nevada State Athletic Commission. Barnett was then given a title shot for the UFC Heavyweight Championship against then-champion and Future UFC Hall of Famer Randy Couture. He won via TKO after using the ground and pound technique and became the new and youngest-ever UFC Heavyweight Champion. However, after the bout it was revealed that he had again tested positive for banned substances and his title was stripped.

Barnett began his overseas career as a professional Wrestler in the New Japan Pro-Wrestling (NJPW) organization, where he wrestled numerous matches in 2003 and 2004. In his first match, he wrestled champion Yuji Nagata for that promotions version of the IWGP Heavyweight Championship. Although unsuccessful in that match, Barnett formed a tag team with Perry Saturn and was undefeated in the following NJPW tour, Fighting Spirit 2003. Barnett wrestled over 50 matches in total for the NJPW promotion.

Next, Barnett fought the Polish Judo gold medalist Pawel Nastula at PRIDE 32, the organization's first show in the United States. To fight again in Nevada, the Nevada State Athletic Commission required that Barnett pass a mandatory drug test. In a surprisingly competitive match, Nastula controlled the first round and most of the second. Barnett reversed Nastula from the bottom and was able to secure a toe-hold submission, which earned him the victory. At a post-fight press conference, Barnett complimented Nastula on his performance. Nastula, however, subsequently tested positive for steroids. Barnett then lost a rematch by unanimous decision to Antônio Rodrigo Nogueira at Pride Shockwave 2006.

Barnett debuted in Antonio Inoki's Inoki Genome Federation (IGF) wrestling promotion in June 2007 with a victory over Tadao Yasuda. He racked up victories against Don Frye and Montanha Silva before suffering his first loss against Naoya Ogawa. The same man he beat earlier in the night. Since the loss, he has been on a winning streak beating The Predator, Hitokui Yoshiki, Tank Abbott, Jon Andersen, Fonseca, Atsushi Sawada, Bob Sapp, Ultimate Mask, Tim Sylvia, Montanha Silva, Bobby Lashley and Hideki Suzuki.

Since his contract with World Victory Road is not exclusive, Barnett was able to join other MMA events. Barnett participated in the inaugural MMA event held by Affliction Entertainment, in July 2008. Seven years after his only knockout loss to Pedro Rizzo, he avenged that loss at Affliction: Banned with a knockout of his own in the second round.

Barnett competed in the California Classic 2009 BJJ Tournament on November 15, 2009. Barnett lost a decision to ADCC veteran and Brazilian jiu-jitsu World Champion Romulo Barral from Gracie Barra in the Black Belt GI Absolute division. On December 19, 2009, Paulson awarded Barnett his Brazilian jiu-jitsu black belt. Even though Barnett had never trained in Brazilian jiu-jitsu he was given his belt based on competition merit and overall knowledge.

On September 13, 2010 it was announced that Josh Barnett had signed a multi-fight deal with Strikeforce.

Starting in February at IGF Genome 14, IGF began a title tournament to crown a new Heavyweight Champion and Barnett not only continued his winning streak but he advanced to the semi-finals of the tournament with his victory over Montanha Silva. In July at IGF Genome 16, he defeated Bobby Lashley with a cross armbreaker to advance to the finals of the title tournament. Was scheduled to face Jérôme Le Banner for the IGF Heavyweight Championship in a tournament final scheduled for August 27 at the IGF Super Stars Festival 2011. However, it was announced on August 19 that Barnett had to pull out of the match up and Le Banner was declared the first ever IGF champion on August 22. However, Josh Barnett would get his shot at the IGF Heavyweight Championship on December 2 against the champion, Jerome Le Banner. Barnett would lose the contest by knockout.

He faced fellow finalist Daniel Cormier on May 19, 2012 at Strikeforce: Barnett vs. Cormier to determine the Strikeforce Heavyweight Grand Prix Champion. Dana White announced if Barnett were to defeat Cormier, he would be allowed back into the UFC, "If he wins the fight, I can't see why he wouldn't come [to the UFC]." Barnett lost the bout via unanimous decision (49-46, 50-45, 50-45), losing for the first time in over 5 years. In January 2013, Barnett faced a promotional newcomer, Nandor Guelmino, at the final Strikeforce event. Barnett defeated Guelmino via arm-triangle choke submission in the first round.

Barnett appeared as himself in an episode of the 2013 web series Black Dynamite Teaches a Hard Way!, where a Black Dynamite mannequin teaches him the consequences of littering.

Barnett has also done his share of acting, appearing alongside Steven Seagal in 2015's Absolution and Michael Jai White in 2016's Never Back Down: No Surrender.

In December 2016, UFC was notified of a potential USADA doping violation by Barnett from an out-of-competition test. On March 2018, Barnett was clear to fight and received a "public reprimand" instead of a suspension from USADA after it was determined that his failed test was the result of a contaminated supplement.

Barnett returned to IGF on April 5th 2017, defeating Shinichi Suzukawa on the promotion's inaugural Next Exciting Wrestling (NEW) event.