The game opens as the Pillar of Autumn exits slipspace near a mysterious ring-shaped space station, called "Halo" by the enemy of the game, the Covenant. A Covenant fleet attacks and heavily damages the Pillar of Autumn. Captain Keyes initiates "The Cole Protocol," a procedure designed to prevent the Covenant from learning the location of Earth. While Keyes prepares to land the ship on Halo, Master Chief and Cortana escape via an escape pod, which crash lands on the ring. Cortana and the Chief are the only ones who survive the impact of the escape pod crash.

Keyes survives the Pillar of Autumn '​s crash landing, but is captured by the Covenant. In the second and third levels of the game, Master Chief and Cortana gather human survivors and rescue Captain Keyes, who is imprisoned aboard the Covenant ship Truth and Reconciliation. Once rescued, Keyes orders Master Chief to beat the Covenant to Halo's control center and to discover its purpose. Master Chief and Cortana travel to a map room called the Silent Cartographer, which leads them to the control room. There, Cortana enters the systems and, discovering something urgent, suddenly sends Master Chief to find Captain Keyes while she stays behind. While searching for his commander, Master Chief learns that the Covenant have accidentally released the Flood, a parasitic alien race capable of spreading itself by overwhelming and infesting other sentient life-forms. Keyes falls victim to them while looking for a cache of weapons. The release of the Flood prompts Halo's AI monitor, 343 Guilty Spark, to recruit Master Chief in retrieving the Index, a device that will activate Halo and prevent the Flood from spreading beyond the facility.

After Master Chief retrieves and prepares to use the Index, Cortana re-appears and warns him against the activation. She has discovered that Halo's defense system is a weapon designed to kill all sentient life in the galaxy, which the Flood requires to spread, a revelation which Guilty Spark confirms.

Faced with this information, Master Chief and Cortana decide to destroy Halo to prevent its activation. While fighting the Flood, the Covenant, and Guilty Spark's Sentinels, Cortana discovers that the best way to accomplish this is to cause the crashed Pillar of Autumn to self-destruct. However, Captain Keyes' authorization is required to destroy the Autumn, forcing the Chief and Cortana to return to the now-Flood-infested Truth and Reconciliation to search for him. By the time that they reach Keyes, however, he has been infected beyond the point of no return by the Flood. Master Chief retrieves Keyes' neural implants directly from his brain and retreats to the Autumn, where Cortana activates the ship's self-destruct sequence. However, 343 Guilty Spark reappears and deactivates the countdown, discovering the record of human history in the process. Master Chief manually destabilizes the Pillar of Autumn's fusion reactors, and he and Cortana narrowly escape the destruction of the ring via a Longsword.

In a brief post-credit scene, 343 Guilty Spark is seen fleeing Halo's destruction and into the unknown of deep space.