• James T. Kirk: William Shatner.
• Spock: Leonard Nimoy.
• Leonard "Bones" McCoy: Deforest Kelley.
• Chekov: Walter Koenig.
• Sulu: George Takei.
• Uhura: Nichelle Nichols.
• William Decker: Stephen Collins
• Ilia:Persis Khambatta
• Klingon: Mark Lenard

Two and a half years after the end of the mission of Kirk (who has now become an admiral) and his crew, the Enterprise has been refitted inside and out, almost an entirely new ship, and some of the crew have drifted apart - McCoy has taken an extended leave, Kirk has accepted a desk job, and Spock has returned to Vulcan to pursue the Kolinahr discipline, a total purge of emotions. In the meantime, Sulu and Uhura have stayed with the Enterprise during its testing phase, while Chekov has become ship's chief of security and Nurse Chapel has become a full doctor.

Captain Willard Decker, son of the late Matt Decker, is slated to become the ship's new commanding officer. An "energy cloud" of unknown origin and intent has carved a path of destruction through the galaxy on a direct course for Earth, having destroyed a flotilla of Klingon ships as well as Federation communications relay station Epsilon 9. Admiral Kirk convinces Starfleet to give him command of the Enterprise, displacing Decker to the position of first officer. The refitted ship still has problems, most notably a transporter malfunction which kills two incoming crew members, including the ship's new Vulcan science officer, whose duties Kirk again hands to Decker. Once the transporter is repaired, the final crew members board the Enterprise, such as Lt. Ilia, the ship's new navigator who once had a relationship with Decker on her home planet of Delta IV; and Dr. McCoy reluctantly resumes his position after being called back into service by Starfleet.

Kirk's unfamiliarity with the Enterprise's new design is proven when he orders the ship to warp speed against the recommendations of Decker and Scotty, plunging the ship into a wormhole which it escapes with a last minute order from Decker. While repairing the damage, the ship is boarded by a ship from Vulcan carrying Spock, who offers to resume his post as science officer. Spock begins by helping Scotty overcome the difficulties with the warp engines, enabling the Enterprise to head for the cloud at top speed. En route, Spock reveals that he was unable to complete his Kolinahr training because he detected an intelligence which he believes is part of the cloud.

Penetrating the cloud, the Enterprise wards off an attack but is weakened in the process. After Spock manages to devise a makeshift message to speak to the cloud-entity in its own language and frequency, the ship delves further into the cloud and is boarded by a beam of energy which tries to access the ship's records on Starfleet and Earth defenses. Spock damages the computer so the beam cannot gather any more information, but is attacked by the beam, which then seems to envelop Lt. Ilia and disappears from the ship, leaving no trace of Ilia. The Enterprise is trapped inside an enclosed, solid space within the cloud, and Ilia turns up again soon afterward, but this time as a puppet of the cloud-entity, identified by the now-dehumanized Ilia as V'ger. Curious to find more about V'ger, Spock steals a space suit and a thruster pack and launches himself into a small opening through which the Enterprise cannot travel, and finds himself floating through the memories of V'ger's entire journey through the universe, eventually coming to an image of Ilia as she was before V'ger's invasion of the bridge. Spock tries to mind-meld with V'ger through the image, but the staggering amounts of V'ger's memory and thought overloads Spock's mind, and he is ejected back to the Enterprise, where he is recovered and given medical attention. The Ilia-probe tells Kirk that V'ger is on its way to Earth to find its own creator, although V'ger refuses to believe that its creator could be a member of the human race, which it intends to wipe out, if necessary, to complete its search. The cloud has reached Earth and is ready to commence with its task.

When Kirk promises the Ilia-probe that he has the information V'ger seeks, V'ger releases the Enterprise and draws it to the center of the cloud, where V'ger itself rests. Kirk, Spock, McCoy and Decker, led by Ilia, find that V'ger is, in fact, a NASA Voyager space probe that was encountered by a race of intelligent machines and, taking the probe's instructions - to learn all it can and report its findings back its creator - literally, the machines created the cloud-vessel as a means for Voyager to return to Earth and deliver its wealth of information. But the probe is unwilling to transmit its information on command, demanding to become one with its creator. Decker manually forces Voyager to transmit its information, but is absorbed by a wave of energy when V'ger believes its creator - the only being who could operate it - has arrived. Kirk, Spock and McCoy rush back to the Enterprise just in time. The cloud dissipates, leaving the Enterprise in orbit over Earth. Kirk and Spock speculate that Decker's emotions concerning his relationship with Ilia, the loss of his command of the Enterprise, and other feelings will transform V'ger into a new life form that the Federation may meet again in the future.