An entire block behind the ABC late-night show's studio was closed down for her live performance on the Bud Light Outdoor Stage in Hollywood, which was attended by scores of fans.
GaGa, who was making her first appearance on the programme since October 2008, follows in the footsteps of artists including Coldplay, Jay-Z and Depeche Mode who have previously shut down areas of Hollywood during their performances.
GaGa, who was dressed in head to toe black, performed new single 'You And I' first, changing the lyrics at one point to, 'There's something about Jimmy Kimmel, you and I' and 'Hollywood I'd rather die without you and I'.
Kimmel tweeted after the show: "Those who aren't fans of @ladygaga haven't seen her perform live. She is as good as they get."
Watch Lady GaGa performing 'You And I' on Jimmy Kimmel Live! below:
GaGa then performed her previous single 'Edge of Glory', telling Kimmel that she was thinking of the late saxophonist Clarence Clemons, who played on the track and appeared in the music video before his death in June.
She said: "The amazing Clarence Clemons played saxophone on 'Edge of Glory', and God bless him, he's not here but I'm going to dance to the moon for him tonight."
Watch Lady GaGa performing 'Edge of Glory' on 'Jimmy Kimmel Live!' below:
GaGa also gave an interview to Kimmel before her performance, where she spoke about topics including her love of cooking, hopes to have children a "long way" in the future, and recent nomination for induction into the Bowling Hall of Fame.
"I'm a good bowler pre-Budweiser, but once the beers start loading up I become worse and worse and worse which is really great for whoever is taking me on the date because I'm a good, cheap date and a terrible bowler!"
As for whether she hires bowling shoes, she said: "Absolutely not! What if one of my fans walked into the bowling alley and saw me in flats? I think they'd have a heart attack!"
GaGa also performed on last night's So You Think You Can Dance results show, following her role as a guest judge on Wednesday night alongside Rob Marshall.
> Lady GaGa to co-host Hot Topics on The View
> Lady GaGa reveals 'You And I' music video details