Junpei awakens in a cabin on a cruise ship, wearing a bracelet with the number "5". He escapes the room and runs into the other eight passengers. Zero declares over the loudspeaker that all nine are taking part in the Nonary Game. Zero outlines the rules, stating that each character carries an explosive in their stomach that will detonate if they attempt to breach the digital root door locks. The 9th Man is still slain by going through a door by himself. Fearing for their safety, the gang chooses code names and divides into groups to explore the ship. The player can choose which party Junpei travels with, which changes the tale; various options result in Junpei's death. Junpei learns of a prior Nonary Game, performed nine years ago, and the relationships of the other characters through that, as well as studies concerning morphic resonance and stories of the Egyptian priestess Alice, who is frozen in Ice IX, through various options.
Junpei discovers in one ending that the first Nonary Game was managed by Cradle Pharmaceutical, of whom Ace is the CEO. Zero was a participant in this game, and he had set up the second Nonary Game as a form of retaliation against Ace. Ace is confronted by the surviving players, who discover that he forced the 9th Man to break the rules and murder himself in order to conceal his identity and steal the 9th Man's bracelet. Akane grows weakened as they approach the door marked with the number 9. While the others enter the door, which leads to an incinerator, Ace takes Lotus and holds her at gunpoint. Snake attacks Ace as the incinerator is about to activate, and Lotus and Seven get Junpei out of the incinerator before it explodes, engulfing Snake and Ace. Junpei returns to Akane and discovers that she is practically dead. Over the loudspeakers, Zero announces that the game's loser has been selected; Junpei reacts angrily, but Zero clarifies that he is speaking to himself. Junpei examines a neighbouring chamber and returns to discover Akane and Santa gone, only to be knocked out by a gas grenade.  After viewing this ending, the player will be able to reach the "real" ending.
Junpei finds in the genuine ending that the last Nonary Game was made up of nine pairs of kidnapped siblings divided on the ocean-bound Gigantic and in a mock-up at Building Q in the Nevada desert. The game was created to investigate morphic fields; the research predicted that the stress of the game would activate the fields between siblings, allowing solutions solved by one to be communicated to their counterpart at the other place via these fields. Ace's prosopagnosia was being treated with this research. This Nonary Game went awry: instead of being split, Akane and her brother Santa were placed in the same area, and Seven discovered the kidnappings and rescued the children from the ship. Before they could go, Ace seized Akane and dragged her into the incineration chamber, threatening her with death if she didn't complete a riddle. Akane was probably burned to death after failing to complete the problem, while the other children escaped with Seven.
Junpei and the others arrive at the incinerator, where Akane vanishes and Santa flees with Ace as a hostage, keeping the others within. It is then revealed that the portion of the game's narrative displayed on the bottom screen of the Nintendo DS during the first Nonary Game is presented from a 12-year-old Akane's point of view (in the game's 2017 re-release, Akane's perspective is actually the "NVL Mode," which displays narrative text rather than the "ADV Mode"). She connected to Junpei in the future via morphic fields, experiencing numerous alternative worlds and directing Junpei to help him survive. Junpei later encounters the same puzzle that Akane did and sends the solution to Akane in the past, allowing her to flee with Seven and the other children. Junpei discovers that Akane was Zero and that she had manufactured the game and all the events she had experienced with the help of Santa in order to assure her survival and prevent a time paradox. As Junpei and the others flee, they discover that the game had taken place the entire time at Building Q, and that Akane and Santa had departed, leaving behind a car with Ace bound in the trunk. In the game's epilogue, they drive away, expecting to catch up with them, and pick up a hitchhiker named Alice, whom Junpei remembers.