The season's storyline picks up directly after last season's finale.

The season takes place fourteen years after the ninth season (2001–02) and seven years after the film The X-Files: I Want to Believe (2008),and follows newly re-instated FBI agents Fox Mulder and Dana Scully as they learn more about the existence of extraterrestrials and their relationship with the government.

After what happened during the season finale of season eight, Fox Mulder goes into hiding. Dana Scully is again reassigned to the FBI Academy and Monica Reyes becomes John Doggett's new FBI partner at the X-Files office. Doggett asks Scully for help on a case involving an EPA official, Carl Wormus, who died after his car was forced off a bridge by a woman he picked up. Doggett and Walter Skinner travel to a reclamation plant, to find any links between Wormus' work and death. After doing an illegal autopsy on Wormus, Alvin Kersh sends Brad Follmer to locate Doggett. The investigation at the reclamation plant leads to an unknown woman, whose identity is later found to be Shannon McMahon, one of Doggett's former Marine associates. She reveals to Doggett that she is a "Super Soldier". This leads them to a clandestine laboratory where a secret experiment is taking place on board on a naval ship. They later find connections with the experiments on the ship to Scully's child, William.

Hopeful about reuniting with Mulder, a complete stranger offers his service to drive Mulder out of hiding. Scully takes the offer, but unknowingly gets herself and Mulder in even more danger. The "Shadow Man", a government agent, follows Scully. It is then discovered that he is a "Super Soldier" bent on killing Scully and Mulder. As confirmed in this episode, a "Super Soldier"'s only weakness is magnetite, which leads to the death of the "Shadow Man". Later on, Scully, Doggett and Reyes find evidence of a dangerous UFO cult which has found a second spacecraft similar to one Scully studied in Africa two years ago. Misled by the FBI, the agents enlist the help of The Lone Gunmen to protect Scully's son after they learn that the UFO cult apparently intend to kill the child. Doggett is run over by a car, leading him to be sent to the local hospital. As Follmer and the "Toothpick Man" are trying to uncover the plans of the three agents, Scully and Reyes leaves Washington, D.C. to find Scully's son.

Scully meets Special Agent John Doggett, the leader of an FBI task force organized to conduct a search for Mulder. Although the search ultimately proves unsuccessful, Doggett is assigned to the X-files and works with Scully to look for explanations to several cases. When Scully learns that several women have reportedly been abducted and impregnated with alien babies, she begins to question her own pregnancy and fears for her unborn child.

After the events of the season six finale, Walter Skinner and Michael Kritschgau are desperately attempting to find the truth behind the so-called alien object. Meanwhile, Fox Mulder is still imprisoned by his own frenetic brain activity. Dana Scully and Skinner are unaware of FBI Special Agent Diana Fowley's duplicity—she is working for The Smoking Man. Scully then travels to Africa to unravel the secrets of the alien artifacts, finding something that looks like a spaceship buried under the shoreline off the Côte d'Ivoire coast. The object may prove that life originated elsewhere, and all religion is based on the Navajo contact with alien life. Unsuccessful, Scully returns from Africa to revisit Mulder in Washington, D.C., but instead she finds out that he has disappeared. She contacts Kritschgau and Skinner to find her partner. The Smoking Man has taken Mulder to a place where all his problems seem to have disappeared. Fowley helps Scully locate Mulder, which leads to her death at the hands of The Smoking Man.

While investigating a bizarre disappearance of a young girl from her home, Mulder becomes obsessed with the number of children who have vanished in similar circumstances. Scully fears that he is emotionally involved due to his sister's disappearance. At the same time it is revealed to him that his mother, Teena Mulder, committed suicide. He then tries to prove that his mother did not take her own life, but is ultimately forced to accept that his mother's death was by her own hand. He is led by a man whose son disappeared years earlier to another truth—that his sister may be among the souls taken by "walk-ins", saving the souls of children doomed to live unhappy lives. Together they locate evidence that proves that Samantha was abducted by The Smoking Man, and was forced to live in a now-abandoned US Army base. It is later revealed that Samantha had become a "walk-in" spirit.

Mulder suspects demon fetal harvesting when a woman claims her unborn child had a horn and tail and was taken by a devil while she slept. While others conclude that the woman killed her own baby, Mulder suspects that her husband is involved and that he may be the devil in disguise.vMulder suspects demon fetal harvesting when a woman claims her unborn child had a horn and tail and was taken by a devil while she slept. While others conclude that the woman killed her own baby, Mulder suspects that her husband is involved and that he may be the devil in disguise.

Scully is suffering from cancer while Mulder is doubtful that aliens exist. The agents fake Mulder's death in an effort to discover which members of the FBI they can trust before individually searching for an answer to Scully's cancer. Her condition worsens, however, and she is hospitalized before Mulder is offered an alleged cure by the Cigarette-Smoking Man. The man also brings Mulder a woman who claims to be his sister, Samantha, although she leaves with the Cigarette-Smoking Man shortly thereafter. When the man offers Mulder a deal to join him, Mulder immediately refuses. He returns to the FBI Headquarters, where he announces that Section Chief Scott Blevins is responsible for Scully's cancer. Both the Section Chief and the Cigarette-Smoking Man are shot. Scully begins to recover and eventually resumes her work on the X-files with Mulder. While staying with her family at Christmas time, she learns that the adopted daughter of a deceased woman is apparently her own child.

When the Syndicate suspect that one of their members is passing information to Mulder and Scully, they organize a canary trap to find the leak, using information about the safety of Mulder's mother as bait. X's role as an informant is discovered, and he is shot dead, although he is able to pass along the name of another informant who can be of use to Mulder—Marita Covarrubias, the Special Representative to the Secretary-General of the United Nations. Covarrubias' aid is sought when Mulder attempts to reach Tunguska in Russia to investigate the source of a further black oil contamination. Whilst there, Mulder is held in a gulag and used as a successful test subject for a black oil vaccine. He escapes and is able to return to America, having found that Alex Krycek is working with the Russians.

Having been diagnosed with cancer, Scully is unsure of her future with the FBI. Mulder is convinced that her condition is a result of her earlier abduction, and is prepared to make a deal with the Syndicate to find a cure. He is dissuaded by Walter Skinner, who secretly makes such a deal instead. While being pursued by an assassin responsible for a hoax alien corpse discovered on a mountaintop, Mulder fakes his own suicide, mutilating the assassin's face to provide a decoy body. He uses the distraction this offers to infiltrate The Pentagon to find a cure for Scully's cancer, while Scully is able to uncover and reveal a Syndicate connection within the FBI.

Investigating evidence of an alien autopsy, Mulder infiltrates a secretive government train carriage carrying an alien-human hybrid. Mulder is almost killed by a Syndicate operative guarding the hybrid, but is saved by his informant X. X had been tipped off about Mulder's activities by the agent's partner Dana Scully. Scully, meanwhile, meets a group of women with abduction experiences similar to her own, and meets another member of the Syndicate known as the First Elder, who claims during her abduction she was placed on a similar train car and experimented upon by the Japanese scientists. The crew of a French salvage ship trying to raise a World War II-era submarine from the sea floor are stricken with massive radiation burns—except for one, who has been infected with a parasitic black oil discovered on the submarine. The oil is controlling the crewman's body, and after passing through several hosts, has overtaken Alex Krycek, who Mulder has been pursuing. Scully finds that the submarine had been involved in discovering the oil on the sea floor during World War II, under the guise of finding a sunken fighter plane. The infected Krycek makes his way to a missile silo used to hide a UFO, and the oil escapes his body to board the craft. Meanwhile, Scully has tracked down Luis Cardinal, the man responsible for killing her sister.

The X-Files are no more. Shut down by the FBI, Scully and Mulder have been reassigned. New supernatural events prove that the X-Files investigations are a necessity and Walter Skinner restores them but with a new team, Fox Mulder and Alex Krycek. Mental patient and alien abductee Duane Barry kidnaps Scully and takes her to a mountain where she disappears. Krycek murders Barry. He works for the Cigarette Smoking Man and disappears with his true loyalties revealed. Scully is returned on the edge of death and Mulder faces a critical choice, seek to ambush members of the conspiracy with the help of his new source Mr. X or stay by Scully's bedside. He chooses Scully. Scully recovers and rejoins Mulder on the X-Files.