In 1989, seven years after the events of the first film, Kevin Flynn, who has been recently promoted CEO of ENCOM International, disappears. Twenty years later, his son Sam, now ENCOM's primary shareholder, takes little interest in the company beyond playing an annual prank on its board of directors.

After being freed from police custody for his most recent prank, Sam is asked by Alan Bradley, an ENCOM executive and Flynn's old friend, to investigate a strange pager message originating from Flynn's shuttered video arcade. Sam discovers a large computer in a hidden basement, which suddenly teleports him to the Grid, a virtual reality created by Flynn that exists within ENCOM’s computer mainframe. He is quickly captured and sent to "the Games", where he is forced to fight a masked program named Rinzler. When Sam is injured and begins bleeding, Rinzler realizes that Sam is a human "User" and takes him before Clu, the Grid's corrupt ruling program who resembles a younger Kevin Flynn. Clu nearly kills Sam in a Light Cycle match, but Sam is rescued by Quorra, an "apprentice" of Flynn, who conveys him to his father's hideout outside Clu's territory.

Flynn reveals to Sam that he had been working to create a "perfect" computer system and had appointed Clu and Tron (a security program created by Bradley) its co-creators. During this construction, the trio discovered a species of naturally occurring "isomorphic algorithms" (ISOs) not conceived by Flynn, bearing the potential to resolve various mysteries in science, religion and medicine. Clu, considering them an aberration, betrayed Flynn, seemingly killed Tron, and destroyed the ISOs. Meanwhile, the "Portal" permitting travel between the two worlds had closed, leaving Flynn trapped in the system. Having gained complete control, Clu sent the message to Alan in order to lure Sam onto the Grid and reopen the Portal for a limited time. As Flynn's "identity disc" is the master key to the Grid and the only way to traverse the Portal, Clu expects Sam to bring Flynn to the Portal so he can take Flynn's disc, go through the Portal himself, and impose his idea of perfection on the human world.

Against his father's wishes, Sam returns to Clu's territory on Quorra's tip-off to find Zuse, a program who can provide safe passage to the Portal. At the End of Line Club, its owner Castor reveals himself to be Zuse, then betrays Sam to Clu's guards. In the resulting fight, Flynn rescues his son, but Quorra is injured and Zuse gains possession of Flynn's disc. Zuse attempts to bargain with Clu for the disc, but Clu simply takes the disc and destroys the club along with Zuse. Flynn and Sam stow away aboard a "solar sailer" transport program, where Flynn restores Quorra and reveals her to be the last surviving ISO.

The transport is intercepted by Clu's warship; as a diversion, Quorra allows herself to be captured by Rinzler, whom Flynn recognizes as Tron, reprogrammed by Clu. Sam reclaims Flynn's disc and rescues Quorra, while Flynn takes control of a Light Fighter on the flight deck. Clu, Rinzler, and several guards pursue the trio in Light Jets. Upon making eye contact with Flynn, Rinzler remembers his past and deliberately collides with Clu's Light Jet, but Clu uses Tron's spare baton to escape while Tron falls into the Sea of Simulation below. Clu confronts the others at the Portal, where Flynn reintegrates with his digital duplicate, destroying Clu along with himself. Quorra, having switched discs with Flynn, gives Flynn's disc to Sam and they escape together to the real world. In Flynn's arcade, Sam backs up and deactivates the system. He then finds a waiting Bradley and tells him he plans to retake control of ENCOM, naming Bradley chairman of the board. He departs on his motorcycle with Quorra, and she witnesses her first real sunrise.

• Jeff Bridges as Kevin Flynn, the former CEO of ENCOM International and creator of the popular arcade game Tron based on his own experiences in ENCOM's virtual reality, who disappeared in 1989 while developing "a digital frontier that will reshape the human condition".

• Bridges also portrays Clu (Codified Likeness Utility), a more advanced incarnation of Flynn's original computer-hacking program, designed as an "exact duplicate of himself" within the Grid, via digital makeup and voiceover, while John Reardon portrays Clu physically.

• Garrett Hedlund as Samuel "Sam" Flynn, a primary shareholder of ENCOM who, while investigating his father's disappearance, is transported onto the Grid himself. Hedlund won a "Darwinian casting process" which tested hundreds of actors, being chosen for having the "unique combination of intelligence, wit, humor, look and physicality" that the producers were looking for in Flynn's son. The actor trained hard to do his own stunts, which included jumping over cars and copious wire and harness work. Owen Best portrays the seven-year-old Sam Flynn.
• Olivia Wilde as Quorra, an "isomorphic algorithm", adept warrior, and confidante of Kevin Flynn in the Grid. Flynn refers to her as his "apprentice" and has imparted volumes of information to her regarding the world outside of the Grid, which she longs to experience. She is shown to have a love of human literature, particularly the writings of Jules Verne, and plays Go with Flynn. She comments that her 'aggressive style' is usually foiled by Flynn's patience. Wilde describes Quorra as akin to Joan of Arc. Her hairstyle was influenced by singer Karen O. Wilde added that although " could have just been another slinky, vampy temptress", it was important for her to appeal to both men and women, and that character tried to avoid the typical female lead by having a naiveté and childlike innocence adequate for such an "evolving and learning organism". Quorra's action scenes led Wilde to work out and train in martial arts.
• Bruce Boxleitner as Alan Bradley, an executive consultant for ENCOM, and close friend of Kevin Flynn who, after receiving a cryptic page from the office at the shuttered Flynn's Arcade, encourages Sam to investigate its origin.

• Boxleitner also portrays Tron/Rinzler, a security program originally developed by Bradley to monitor ENCOM's Master Control Program and later reassigned by Flynn to defend the Grid, who was overpowered and re-purposed by Clu as a masked command program wielding an identity disk that splits into two, in flashback sequences via the same treatment as Bridges' younger self for Clu. Anis Cheurfa, a stunt actor, portrayed Rinzler, while Boxleitner provided the dialogue. Rinzler is named after author and Lucasfilm Executive Editor J.W. Rinzler.

• Michael Sheen as Zuse/Castor, a flamboyant supermodel program who runs the End of Line Club at the top of the tallest tower in the system. Sheen describes his performance as containing elements of performers such as David Bowie, Joel Grey from Cabaret, and a bit of Frank-N-Furter from The Rocky Horror Show.
• James Frain as Jarvis, an administration program who serves as Clu's right-hand man and chief intelligence officer. Frain had to shave his head, bleach his eyebrows white, and wear make-up. The refraction on Jarvis' helmet led Frain to walk in a "slightly squinty, blind stagger" which the actor felt was helpful to get him into character. Frain described Jarvis as "a fun, comic character that’s a little off-