Portal 2 will continue to challenge the player by solving puzzles in test chambers within the Aperture Science Enrichment Center using the portal gun (the Aperture Science Hand-held Portal Device), a device that can create two portals connecting two surfaces across space. Players, as the silent protagonist Chell from Portal, solve puzzles by using these portals to move unconventionally between rooms or to use the ability to fling objects or themselves across a distance. The functionality of the gun has not changed between the games, but within Portal 2, players can take advantage of the bleeding of other physical effects through the portals. Game Informer identified two examples of this: one was the ability to use air currents created by Pneumatic Diversity Vents, a series of transport pneumatic tubes, through a set of portal openings to push a turret over or to draw objects into the suction. The second example was to use the power of Excursion Funnel tractor beams through portals to bring Chell or other objects to otherwise inaccessible areas. The game also introduces special paint-like gels that can be used to impart certain physical effects to a surface such as Propulsion Gel that boosts Chell's speed as she crosses a surface, and Repulsion Gel that allows her to jump from a surface. The player will be required to determine how to transport that gel to appropriate surfaces using portals in order to progress. The gels can also be applied to objects, such as the Weighted Storage Cube crates, that affect their own physical nature. In addition to the Storage Cube, there are new types of portable objects that assist the player, including Redirection Cubes with mirrored, reflective surfaces used to redirect Thermal Discouragement laser beams, Aerial Faith Plates that can launch objects placed on them, and spherical Weighted Storage Balls, which made a brief appearance in the original game in one of the advanced chambers.

While most of the single-player game will take place in the test chambers created by GLaDOS or her personality cores, there will be times where the player will need to move behind-the-scenes in areas beyond the test chambers as they are reconfigured, leaving the player free of GLaDOS's observation and control.

The game will include a two-player co-operative mode in addition to the single player mode. Both players control separate portal guns and can use the other player's portals as necessary; the second player's portals will be of a different color to help distinguish between the two sets. Because of the number of possible portal combinations, the test chambers that the players proceed through are much more difficult than the single-player campaign, encouraging the two players to work together. This includes complicated laser redirection using both series of portals, or working in two separate chambers, performing actions in their side of the chamber to allow the other player to progress in theirs. The game includes voice communication between players for this mode as well as split-screen for players playing locally. Online players will have the ability to temporarily enter a split-screen view to help coordinate actions. Players have the ability to place icons on the game's levels as a means of informing the other player what they need to do

<b><big>Plot</big></b>
Portal 2 takes place hundreds of years after the first game. Despite her apparent destruction at the end of Portal, GLaDOS is "still alive". The player controls Chell, the same protagonist from Portal; retroactively patched just prior to the sequel's official announcement, the ending of the first game shows Chell being dragged away by an unseen figure with a robotic voice, where she has been placed in stasis over the years. The game will again take place in the Aperture Science Labs, untouched by human hands but overrun by decay and nature. The player will interact with many of the numerous personality cores (which were seen activating in Portal's conclusion), which have become active and become independent from GLaDOS in the intervening years, using the automated systems of Aperture Science to create their own microcosms within the facility. The cores themselves are unable to move save through overhead rail systems. Chell is awoken by one of these, Wheatley, who has become concerned for the state of decay and seeks to correct it. Wheatley acts as the player's guide during the tutorial and initial stages. Soon, the two encounter the dormant GLaDOS and accidentally wake her; the computer accuses Chell of murdering her years ago. GLaDOS begins to rebuild the ruined facility, and puts Chell under more tests, stating "I think we can put our differences behind us. For science. You monster."

Two new characters will be introduced for the two-player cooperative mode, which will have its own unique plot and setting. These two yet-to-be-named characters include a modified turret gun and a personality core; both units are bipedal and equipped with their own portal guns. Though once part of the networked facility, they have become separate entities and are treated to similar abuse by GLaDOS while Chell is being put through a series of complicated test chambers.