Stargate Atlantis follows the present-day adventures of Major John Sheppard and his military team from Earth that, along with two dozen other teams, explore distant planets in the Pegasus Galaxy. They use an alien device known as a Stargate that was built millions of years ago by an advanced race of humans known as the Ancients. The expedition is based in the Lost City of Atlantis on the planet "Lantea". The city was built millions of years ago and abandoned 10,000 years ago by the Ancients. Five to ten million years ago, due to a plague in the Milky Way Galaxy, they were forced to flee to the Pegasus Galaxy, and there they seeded life on hundreds of worlds as they had done to Earth in the Milky Way. After encountering a powerful enemy known as the Wraith and going to war with them for one hundred years, the Ancients ultimately lost and were forced to submerge their city beneath Lantea's ocean, which, in the Stargate universe, is the source of the Greek myth of the Lost City of Atlantis.

Stargate: Atlantis episodes feature a self-contained story that also contributes to the larger storyline of the war against the Wraith and the Atlantis residents' search for the means to destroy their enemy. Each season has also featured a two-part episode, and some episodes that, while not technically two-parters, feature direct continuity with the story of the previous episode (for example, season 3 "Progeny" and "The Real World"). Each episode begins with a cold open, sometimes preceded by a recap of events relevant to the upcoming narrative. The opening credits feature an original theme by Joel Goldsmith. Though they were cut at the start of season 2, the full credits were recovered after the mid-season two-parter. They were again cut short in the 5th season.