The space agency announced the news on Tuesday, saying her retirement took effect at the end of December.
Williams' crewmate on Boeing's ill-fated capsule test flight, Butch Wilmore, left NASA last year.
The pair launched to the space station in 2024, the first people to fly Boeing's new Starliner crew capsule. Their mission should have lasted just a week, but stretched to more than nine months because of Starliner trouble. In the end, they caught a ride home last March with SpaceX.
Boeing's next Starliner mission will carry cargo - not people - to the space station. NASA wants to make sure all of the capsule's thruster and other issues are solved before putting anyone on board. The trial run will take place later this year.
Williams, 60, a former Navy captain, spent more than 27 years at NASA, logging 608 days in space over three station missions. She also set a record for the most spacewalking time by a woman: 62 hours during nine excursions.
NASA's new administrator Jared Isaacman called her "a trailblazer in human spaceflight".
"Congratulations on your well-deserved retirement," he said in a statement.
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