Souza made his mixed martial arts debut on September 9, 2003 at Jungle Fight's inaugural event where he lost to Jorge "Macaco" Patino by KO at 3:13 into the first round. He returned to mixed martial arts eight months later at Jungle Fight 2 securing a submission win over Victor Babkir under a minute into the first round. His next fight was in April 2006 at Jungle Fight 6 where he got his second win, defeating Alexander Shlemenko by an arm triangle choke submission. He went on to fight twice before the end of 2006, winning both fights by submission during the first round.

In 2006, Souza fought and drew with UFC Hall of Famer Randy Couture in a grappling contest. After the match Souza was invited by Couture to train at his gym. Souza accepted and started training at Xtreme Couture in Las Vegas. Souza's first fight of 2007 was on May 19 with him scoring a submission win due to strikes over veteran Bill Vucick at Gracie Fighting Championships: Evolution. He would next fight twice within a two-week period on September 29 and October 13, scoring two submission wins both within the first round.

In 2008, Souza signed with Japanese MMA-promotion DREAM and was scheduled to participate in the 2008 DREAM Middleweight Grand-Prix. In the first round of the tournament, at DREAM 2 on April 29, 2008, he defeated Ian Murphy by a rear-naked choke submission in the first round. This secured him a place in the quarter-finals which took place at DREAM 4 on June 15 where he fought former ICON Sport Middleweight Champion Jason "Mayhem" Miller. The fight went the distance with Souza continuously going for submissions with Miller escaping multiple rear-naked choke, leglock and armbar-attempts while mounting his own offense on the feet. Souza was awarded a unanimous decision victory which secured him a place in the semi-final of the Middleweight Grand-Prix.

Souza made his Strikeforce debut on December 19, 2009 at Strikeforce: Evolution with a first-round submission win over Matt Lindland, via arm-triangle choke. In this fight, Souza showed a major improvement in his stand up game and striking. Souza faced Joey Villaseñor on May 15, 2010 at Strikeforce: Heavy Artillery. He won the fight via unanimous decision, dominating the first round, being very aggressive, achieving the mount position and almost achieving the submission, but slowed down on the 2nd and 3rd round.

Souza fought Tim Kennedy on August 21, 2010 at Strikeforce: Houston for the vacant Strikeforce Middleweight Championship, and won via unanimous decision. Souza then defended the belt for the first time against Robbie Lawler, on January 29, 2011 at Strikeforce: Diaz vs. Cyborg.

On August 18, 2012, Souza faced Derek Brunson in his seventh Strikeforce appearance. Souza dropped Brunson early with a counter right hand which planted Brunson on the mat and landed three ground strikes, knocking out Brunson in just 41 seconds.

Souza next knocked out Yushin Okami in the first round on September 4, 2013, at UFC Fight Night 28.

Souza was expected to rematch Gegard Mousasi on August 2, 2014 at UFC 176. When the event was cancelled, the bout was rescheduled for September 5, 2014, at UFC Fight Night 50. Souza won via submission in the third round, earning his first Performance of the Night bonus award.

The bout with Romero was scheduled for a third time, eventually taking place on December 12, 2015 at UFC 194. Souza lost the bout via split decision. 12 of 17 media outlets scored the bout in favor of Souza, and 3 of 17 scored it a draw.

Souza was expected to face Luke Rockhold in a rematch on November 27, 2016 at UFC Fight Night 101. However the pairing was cancelled on 1 November after Rockhold was ruled out of the contest after sustaining an undisclosed injury. As a result, Souza was removed from the card as well.

Souza fought Robert Whittaker on April 15, 2017 at UFC on Fox 24. He lost by second-round TKO. During the event weekend, Souza signed a new, eight-fight deal with UFC.

Souza is scheduled to face Kelvin Gastelum on May 12, 2018 at UFC 224.