The musicians sang part of their 2000 hit "Bye, Bye, Bye" at the Barclays Center in Brooklyn Sunday night.
The reunion followed Timberlake's medley of solo songs, which included "Take Back the Night," "Sexy Back," "My Love," "Cry Me a River," "Senorita," "What Goes Around Comes Around" and "Rock Your Body," and continued after Chasez, Kirkpatrick, Fatone and Bass left the stage.
Comedian Jimmy Fallon then presented Timberlake, 32, with the MTV Video Vanguard Award.
"Most of all, I owe [this award] to those four guys up there," Timberlake said, referring to his former band mates. "Above all else, I'm gonna share it -- I'm gonna keep it at my house, though."
The reunion was the group's first public performance since the 2003 Grammy Awards.
Timberlake also won Sunday night's top prize -- Video of the Year for "Mirrors," which took the trophy for Best Editing, as well.
"Suit & Tie," his collaboration with rapper Jay Z, won the award for Best Direction.