(Commander Shepard can be either male or female, depending on the player's choices. This article will refer to Shepard as a male)

The game begins aboard the experimental SSV Normandy, commanded by Captain Anderson and his executive officer, Commander Shepard, the playable character. The Normandy is being sent to the human colony world of Eden Prime to recover an unearthed Prothean beacon. To assist in recovering the beacon, the Citadel Council, the main government for most of the galaxy, have sent one of their top agents, a turian "Spectre," named Nihlus. Nihlus is also there to evaluate Shepard to see if he is fit to become the first human Spectre.

Nihlus, Shepard, and a small team discover that the Eden Prime colony is under attack by alien synthetic life-forms known as the geth. Nihlus encounters and is killed by a rogue turian Spectre named Saren Arterius, who is working with the geth. After the battle ends, Shepard discovers the beacon, but it suddenly activates. Shepard receives a vision of biological creatures being slaughtered by machines before the beacon explodes.

The Normandy and its crew are summoned by Ambassador Udina, Earth's representative to the galaxy, to the Citadel to report. Shepard is unable to convince the Citadel Council of Saren's treason until he discovers a recording of a conversation between Saren and one of his allies, an asari Matriarch named Benezia, discussing their victory on Eden Prime. The recording also mentions the return of a force known as the "Reapers," as well as an artifact called the "Conduit." Confronted with this evidence, the Council revokes Saren's status as a Spectre, and makes Shepard the first human Spectre so that he legally can hunt down Saren.

Command of the Normandy is taken from Captain Anderson, and given to Shepard. He uses the ship to follow up on several leads provided by Anderson and Udina, but as a Spectre, Shepard is free to follow them in any order. On Therum in the Artemis Tau Cluster, Shepard finds and rescues Dr. Liara T'soni, Matriarch Benezia's daughter, who is an expert on the Protheans. On Feros, Shepard fights off an army of geth and destroys an ancient life form known as the Thorian, a sentient plant-like creature, and also learns of Saren's flagship, Sovereign, which seems to be capable of unique mind-control capabilities. On Noveria, Shepard tracks down Matriarch Benezia while fighting off both geth and alien insects known as rachni. Benezia attacks Shepard and is defeated, and she reveals that her mind is being controlled by Saren, through his ship, Sovereign. She soon reverts to Saren's control, and Shepard is forced to kill her.

After completing two of the previous three planets, Shepard learns a salarian spy unit has uncovered a base operated by Saren on the planet Virmire. Shepard assaults the base to detonate a nuclear device, and inside, he discovers another Prothean beacon, which transfers its knowledge to Shepard. Shepard is then confronted by Sovereign itself, which reveals that it is what the Protheans and geth consider to be a "Reaper." Sovereign explains that the Reapers permit organic life to develop, allowing species to discover the mass relays (which influence future technology) before exterminating them when they reach a certain level of advancement. After planting the nuclear bomb and attempting to escape the facility, the team is attacked by Saren. He explains that he has aligned himself with the Reapers to save organic life by making it "useful" to the Reapers, working as slaves. Shepard refuses to accept Saren's plan, and escapes Virmire before the nuclear warhead is detonated, but at the cost of one of his team members.

With the information Shepard's party has gained, Liara is able to pinpoint the Conduit's location on a Prothean world known as Ilos. Returning to the Citadel with this information, Shepard finds that the Council does not accept the Reapers as a genuine threat. Ambassador Udina locks Shepard out of the Normandy, but Captain Anderson steps in and allows Shepard to steal the ship back.

On the surface of Ilos, Shepard follows Saren into an ancient bunker deep within the planet, and encounters a Prothean computer system named Vigil, which explains the Reapers' methodology. The Citadel Station is actually a mass relay facility built by the Reapers intended to summon them from the dark space outside the galaxy. Before the Reapers invaded, the researchers at Ilos were on the verge of understanding the mass relay system, and were able to finish their studies upon reawakening from cold sleep. The Prothean researchers sabotaged the mass relay in the Citadel and created the Conduit, a miniaturized mass relay that allowed them to travel to the heart of the Citadel. Sovereign, a Reaper that was left behind to re-start the chain of extinction, intends to use the Conduit to transport Saren and his geth army directly into the Citadel, bypassing its security systems.

Shepard pursues Saren through the Conduit. At the same time, Sovereign and a massive geth fleet assault the Citadel itself, breaking through the Council fleets defending the station. Sovereign lands on the central tower of the Citadel and begins activating the relay, while Saren uses the Citadel's central control systems to close the station around the Reaper to protect it. Shepard fights up the tower to the control center and confronts Saren, either killing him personally or convincing him that he has fallen under Sovereign's indoctrination, prompting Saren to kill himself. The Human Systems Alliance fleet arrives to reinforce the beleaguered Council fleets, while Shepard reopens the Citadel and exposes Sovereign. At this point, Shepard has a choice between ordering the Alliance fleet to save the Council, who are under attack by the geth, or going directly to Sovereign. Saren's corpse suddenly reanimates under Sovereign's direct control, through the use of cybernetic implants, and begins attacking Shepard while the Alliance attacks Sovereign itself. Sovereign is destroyed, and the reanimated Saren corpse disintegrates.

The precise ending of the game depends on several factors, including whether or not Shepard opts to save the Council during the final battle, and whether Shepard has a higher Paragon or Renegade meter. If Shepard saves the Council, they are inspired by Shepard's actions and bravery, and grant humanity a seat on the Council. If the Council is destroyed, the Systems Alliance assumes control, and humanity steps forward to lead the galaxy into the future. Shepard is also allowed to choose between Ambassador Udina or Captain Anderson as the new galactic leader or Council member in the aftermath, but regardless of the choice, Shepard realizes that the Reapers are still a threat, and that they must be stopped.