Halo 3 is a shooter game where players primarily experience gameplay from a first-person perspective. Much of the gameplay takes place on foot, but also includes segments focused on vehicular combat. The balance of weapons and objects in the game was adjusted to better adhere to the "Golden Triangle of Halo": these are weapons, grenades, and melee attacks, which are available to a player in most situations. Players may dual-wield some weapons, forgoing the use of grenades and melee attacks in favor of the combined firepower of two weapons. Many weapons available in previous installments of the series return with minor cosmetic and power alterations. Unlike previous installments, the player's secondary weapon is visible on their player model, holstered or slung across the player's back.

Halo 3 introduces "support weapons", which are cumbersome two-handed weapons that slow the player, but offer greatly increased firepower in return. In addition to weapons, the game contains a new class of gear called equipment; these items have various effects, ranging from defensive screens to shield regeneration and flares. Only one piece of equipment can be carried at a time. The game's vehicular component has been expanded with new drivable and AI-only vehicles.

After the events of the comic tie-in Halo: Uprising, the Master Chief crashes in eastern Africa, where he is found by Johnson and the Arbiter. The Chief, Johnson, and company fight Covenant in the jungle and arrive at a UNSC outpost. Here, Keyes and Lord Hood plan a final effort to stop the Covenant leader, the High Prophet of Truth, from activating a Forerunner artifact uncovered outside the ruins of the city of New Mombasa. The Chief clears anti-air Covenant defenses so Hood can lead the last of Earth's ships against the Prophet, but Truth activates the buried artifact, creating a slipspace portal which he and his followers enter. A Flood-infested ship crash-lands nearby; Elite forces arrive and vitrify Flood-infected areas of Earth, stopping the threat. Following a message Cortana left aboard the Flood ship, the Chief, Arbiter, Elites, Johnson, Keyes and their troops follow Truth through the portal. Joining them is 343 Guilty Spark, who aids the Chief as he has no function to fulfill after the destruction of his ringworld.

Traveling through the portal, the humans and Elites discover an immense artificial structure known as the Ark, far beyond the edges of the Milky Way galaxy. Here, Truth can remotely activate all the Halos. The Flood arrive aboard High Charity in full force, beginning to infest the installation. Truth captures Johnson, as he needs a human to use Forerunner technology. Keyes is killed attempting a rescue, and Johnson is forced to activate the rings. Gravemind forges a truce with the Chief and Arbiter to stop Truth. The Arbiter, Master Chief, and Flood forces arrive and overwhelm Truth's guards, rescuing Johnson and halting the installations' activation. After the Arbiter kills Truth, Gravemind turns on the Chief and Arbiter.