She has more than 5.7 million followers. She overtook Spears over the weekend and posted a video late Saturday on YouTube.
Gaga called her fans her "little monsters" as she thanked them for beginning "my reign as Twitter queen."
Spears, Ashton Kutcher, Ellen DeGeneres and an account run by President Barack Obama's political organization, Organizing for America, all have more than 5 million followers.
The most followed title often shifts rapidly, and fans frequently organize to boost their favorite's tally.