In 1989, Kevin Flynn (Jeff Bridges), the software engineer and CEO of ENCOM International, mysteriously disappears four years after the death of his wife Jordan. Twenty years later, his son, Sam (Garrett Hedlund), now ENCOM's primary shareholder, takes little interest in the company beyond playing an annual practical joke on the board of directors. Sam is visited by his father's friend, ENCOM executive Alan Bradley (Bruce Boxleitner), urging Sam to investigate a message originating from Flynn's abandoned arcade. There, Sam unintentionally teleports himself to the Grid, a virtual reality created by his father.
On the Grid, Sam is sent to compete against a masked program called Rinzler; who, having realized that Sam is a User, takes him to CLU, a duplicate of Kevin Flynn who acts as the Grid's ruler. CLU nearly kills Sam in a Light Cycle match; but Sam is rescued by Quorra (Olivia Wilde), an apprentice of his father's, who conveys him to his father outside CLU's territory. There, 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 Alan Bradley, as its co-creators. In process thereof, Flynn discovered a series of "isomorphic algorithms" (ISOs), which carried the potential to resolve various mysteries in science, religion, and medicine; but CLU, considering these an aberration, betrayed Flynn, captured Tron, and destroyed all of the ISOs, with the sole exception of Quorra. Meanwhile, the "portal" permitting travel between the two worlds has closed, leaving Flynn a captive; whereafter CLU sent the message to Alan, to get Sam.
Against his father's wishes, Sam returns to the Grid to find Zuse, a program promised to provide safe passage to the portal. At the End of Line Club owner Castor (Michael Sheen), reveals himself to be Zuse and betrays him to CLU's guards. In the resulting fight, Flynn rescues his son; Quorra is injured; and Zuse gains possession of Flynn's 'identity disc'. Knowing the disc works as a master key to the Grid, Zuse attempts to bargain with CLU; but CLU simply takes the disc and destroys the club. Flynn and Sam save the injured Quorra and stow away aboard a solar sailer, where Flynn restores Quorra.
Aboard a massive warship, Flynn recognizes Rinzler as Tron, reprogrammed by CLU, while CLU announces his desire to invade the material world. Sam thereafter reclaims Flynn's disc; whereupon CLU, Rinzler, and several guards pursue the protagonists in Light Jets. During the pursuit, Rinzler remembers his former identity and destroys CLU's Light Jet. CLU uses Tron's Light Jet to escape while Tron falls into the Sea of Simulation, where the colored lights on his armor change from orange to blue.
CLU confronts the protagonists at the portal, where after attempting to reason with CLU, Flynn absorbs him, destroying themselves as well as CLU's army and warship, while Sam and Quorra transfer themselves to the real world.
In Flynn's arcade, Sam backs up the system on his USB flash drive, and having deactivated it offers to take control of ENCOM and name Alan chairman of the board. Quorra meets Sam outside, and the two depart on his motorcycle; Sam showing Quorra a sunrise en route.
- 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. He disappeared in 1989 while developing "a digital frontier that will reshape the human condition". Bridges also portrays CLU (Codified Likeness Utility), via digital makeup and voiceover, while John Reardon portrays CLU physically. CLU is a more advanced incarnation of Flynn's original computer-hacking program, designed as an "exact duplicate of himself" within the Grid.
- Garrett Hedlund as Sam Flynn, a primary shareholder of ENCOM International. While investigating his father's disappearance, Sam is transported onto the Grid himself.
- Owen Best plays 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 in person. Wilde describes Quorra as akin to Joan of Arc. Her hairstyle was influenced by singer Karen O. Wilde added that although "[Quorra] could have just been another slinky, vampy temptress", it was important for her to appeal to both men and women. She and Kevin Flynn are depicted as recreational Go players.
- Bruce Boxleitner as Alan Bradley, an executive consultant for ENCOM International, close friend of Kevin Flynn, and a surrogate father figure for Sam. After receiving a cryptic page from the office at the shuttered Flynn's Arcade, he encourages Sam to investigate its origin. Boxleitner also portrays Tron, a security program originally developed by Bradley to monitor ENCOM's Master Control Program, and later reassigned by Flynn to defend the Grid, in flashback sequences via the same treatment as Bridges' younger self for CLU.
- Michael Sheen as Castor/Zuse, a vivacious and renowned program who runs the End of Line Club at the top of a tower in the system. Sheen describes his performance as containing elements of performers such as: Ziggy Stardust; Joel Grey from Cabaret; and a bit of Frank-N-Furter from The Rocky Horror Show.
- Beau Garrett as Gem, one of four programs known as Sirens. The Sirens operate the Grid's game armory, equipping combatants with the armor needed to compete in the games, while also reporting to Castor.
- Daft Punk as DJ programs at Castor's End of Line Club.
- Yaya DaCosta, Serinda Swan, and Elizabeth Mathis as the other three Sirens.
- Steven Lisberger as Shaddix, a bartender in the End of Line club.
- Anis Cheurfa, a stunt actor, portrays the masked warrior Rinzler.
- Jeffrey Nordling as Richard Mackey, chairman of ENCOM board.
- Edie Mirman as the Computer Voice heard throughout the Grid.
Cillian Murphy appears in an uncredited role as Edward Dillinger, Jr., head of the software design team for ENCOM and the son of former ENCOM Senior Executive Ed Dillinger (portrayed by David Warner in the original film). This sequel notably doesn't feature most characters from the original (such as the MCP) and barely features the titular character Tron.