The game is set in the year 2415, after a biological disease has wiped out Earth's population except for those living in Bregna, a walled, protected city-state. The city is ruled by the congress of scientists who discovered the vaccine for the disease. Aeon Flux, a top operative in the underground "Monican" rebellion, is sent on a mission to kill one of Bregna's most influential government leaders, Trevor Goodchild. Following a series of self-discoveries and revelations, Aeon uncovers a world of secrets which makes her doubt her mission and question everything she thought she knew.

The game's storyline attempts to bridge the gap between the TV series and the film by explaining various discrepancies such as the appearance of the jungle outside Bregna and the differences between the movie and TV series versions of Trevor Goodchild. The game's visuals and tone skew toward that of the film, and the look of Aeon in the game is based on Charlize Theron's appearance in the film version; the character is also voiced by her.