In 2320, Jacob Lee (Josh Duhamel) and Max Barrow (Jeff Schine) are contract freight transporters working for the United Jupiter Company (UJC). The two decide to retire after ferrying one last shipment between Europa and the UJC-operated Black Iron Prison on Callisto. Shortly after leaving Black Iron in their ship, the Charon, they are boarded by the Outer Way, a terrorist group purportedly responsible for a massive biological attack on Europa, and the ship crash-lands back on Callisto.

Max is killed, leaving Jacob and Outer Way leader Dani Nakamura (Karen Fukuhara) as the only survivors. They are recovered by Black Iron Prison security head Captain Leon Ferris (Sam Witwer) and incarcerated on the orders of Warden Duncan Cole (James C. Mathis III). After an intake process, Jacob awakens to find the prison overrun by hostile "biophages", inmates afflicted with an unknown disease.

He encounters Elias Porter (Zeke Alton), a prisoner who claims to have an escape plan. After freeing Elias, Jacob fights and leaves Ferris to die at the hands of biophages. Elias convinces Jacob to locate a hacker in the Special Housing Unit to summon a ship for their escape. The hacker is revealed to be Dani, but she refuses to join them.

On their way to the hangar, Jacob and Elias are intercepted by a partially-infected Ferris and are ejected onto Callisto's surface; Elias suffers fatal injuries in the process. Dani appears in a half-track and recovers Elias' memories of Black Iron and decides to assist Jacob as the biophages mutate and become more dangerous. After Dani fails to find evidence of the UJC illegally smuggling bioweapons in the wreckage of Charon, the two make it to the hangar where they summon a ship.

Cole intervenes and shoots down the ship, causing it to crash into and destroy the hangar. With their method of escape gone, Jacob and Dani decide to confront Cole. The two make their way through the ruins of Arcas, Callisto's original colony before it was abandoned, and the Black Iron Prison built over it. They find a lab holding a dead creature that the original Arcas miners unearthed, and learn the biophages originated from larvae that were found in the creature.

Seeing its potential for accelerating evolution, the UJC harvested the creature's larvae and began experimenting with it on humans, causing an outbreak in Arcas; one biophage, Subject Zero, retained his intelligence before the UJC destroyed the colony. After Jacob and Dani fend off an encounter with Ferris, Dani becomes infected. Returning to Black Iron, Jacob is knocked out by a security robot and imprisoned.

Jacob is freed by the prison doctor, Dr. Caitlyn Mahler (Louise Barnes), who reveals that Cole is part of a religious sect called Kallipolis, which is attempting to find a way to advance human evolution. He ordered Mahler to experiment on Black Iron prisoners to find a subject compatible with the alien infection and replicate Subject Zero with a Subject Alpha.

Mahler tells Jacob that Dani can be cured by extracting the Alpha's DNA to synthesize an antidote. When Jacob inquires about the Europa outbreak, Mahler admits that a diluted version of the infection was released; the incident inspired Cole to initiate the Callisto Protocol, causing an outbreak in Black Iron by releasing Mahler's test subjects to create the Alpha.

Mahler provides Jacob access to Dani's memories, where Jacob learns that Dani's sister was killed in the Europa outbreak. He recovers his own memories showing he was aware that the UJC was using his ship to smuggle larva samples, but he ignored this due to the payments he was receiving. Jacob confronts Cole, who pits him against Ferris, now mutated into the Alpha. Jacob defeats Ferris and extracts his DNA.

Cole attempts to convince Jacob to turn over the DNA, asserting its value in safeguarding humanity's survival, but Jacob uses it to cure Dani. Enraged, Cole activates the prison's self-destruct countdown and boasts that he has gathered enough data to carry out the Protocol.

Jacob puts Dani in the last remaining escape pod along with an alien larva sample, giving her the evidence she needs to expose the UJC's experiments. Mahler contacts Jacob and informs him of a possible escape as the self-destruct was temporarily postponed, before he is attacked by Ferris.