The miniseries opens with the following text, during a scene in which the Cylons make their first appearance in over forty years at a remote space station, the Armistice station:

It is revealed that the Cylons have gained the ability to mimic the human form. They proceed to destroy the space station.

The Cylons return from their forty-year absence in an attempt to exterminate the human race, beginning with a sudden, unexpected and simultaneous nuclear bombardment of the Twelve Colonies. The attack is made possible by the Cylons' infiltration of the Colonies' defense mainframe, through a Cylon agent known as Number Six who mimics a human woman both physically and behaviorally. Number Six poses as an agent of a company seeking defense contracts and is able to seduce a scientist working for the Colonial military, Dr. Gaius Baltar. In exchange for his access to government mainframes she helps him design a navigation program used by Colonial warships, covertly creating backdoors in the program. When the Cylons attack, they are able to penetrate software security firewalls, disabling entire fighter squadrons outright and sabotaging vital capital-ship systems. (In one incident, an entire battlestar loses power, enabling the Cylons to destroy it easily).

The computer systems of the Battlestar Galactica are not networked and are unaffected by the sabotage. It was in the process of being decommissioned and converted into a museum to honor its role in the first Cylon war, when it receives news of the attack and learns that fleet headquarters, and a significant portion of the colonial fleet, have been destroyed. Commander Adama, Galactica's commanding officer, assumes command of the fleet after hearing that Admiral Nagala has been killed.

To avoid Cylon forces, Galactica makes a 'faster-than-light' (FTL) jump - an instantaneous teleportation - to Ragnar Anchorage, where it can restock on ammunition. Meanwhile, Colonial Heavy 798, which was carrying Secretary of Education Laura Roslin back to Caprica following Galactica's decommissioning ceremony, learns that Caprica City has been destroyed by the nuclear attack. When all 42 government officials ahead of her in the presidential line of succession fail to check in per emergency procedures, Roslin assumes the presidency and Colonial Heavy 798 becomes Colonial One. In the aftermath of the attack, surviving civilian ships turn to Roslin for leadership. Dr. Baltar manages to escape Caprica when Lt. Karl "Helo" Agathon trades his spot on a Colonial Raptor with him. Karl's partner, Lt. j.g. Sharon Valerii, reluctantly consents and flies back to join Galactica and the ragtag fleet. Predicting another Cylon attack after the civilian vessels are spotted by a Cylon Raider, Captain Lee Adama persuades President Roslin to order those civilian ships with hyperjump capability to rendezvous with Galactica at Ragnar. The agonizing decision is made to hastily escape by leaving behind all those humans in the ships with only sublight engines, including a Geminon Botanical Cruiser carrying a little girl met by the president. Captain Adama's advice is proven correct as the Cylons promptly appear and destroy every ship left behind just as the few FTL ships hyperjump to safety.

Roslin eventually convinces Commander Adama that the war is lost. He finally agrees that the best course of action is to escort the civilian fleet and find a new world on which to rebuild civilization. The stockpiles at Ragnar Anchorage are used to rearm Galactica and resupply the fleet. However, the ships cannot jump safely from the immediate vicinity of the base, which is surrounded by a radioactive gas cloud. The Cylons, more vulnerable to the radiation, besiege the cloud and wait for the fleet to emerge. Adama devises a plan: Galactica holds off the Cylon attacks long enough for the civilian ships to exit the cloud and jump to the Prolmar Sector, then joins them.

Afterwards, during the mass funeral for the dead, Adama lifts his crew's morale by claiming that he knows the location of the legendary thirteenth colony known as Earth. Roslin questions him later and realizes that Adama is not even sure it exists, let alone where it is, but agrees to keep the secret.

In the final scenes of the miniseries, Number Six warns Baltar that Cylon agents, who may even be programmed to believe that they are human, could still be within the fleet. Adama finds a cryptic message in his quarters reading simply, "There are only 12 Cylon models." On Ragnar, a group of humanoid Cylons free Aaron Doral (Galactica's tour guide, who was left behind after Baltar "discovered" he was a Cylon). The last Cylon to enter the room is another copy of Lt. j.g. Sharon Valerii, revealing that the one on the Galactica is a Cylon as well.