The boyband hung out in the studio with the Canadian teen sensation earlier this year, fuelling rumors that they were recording a track for the group's new album Take Me Home.
However, asked by The Sun if a hook-up had been recorded, Harry Styles said: "There is no Justin Bieber on the album."
Liam Payne had previously denied the speculation, explaining that they visited Bieber in the studio to listen to some of his new material.
"We actually went down to the studio to listen to some of Justin Bieber's tracks," Payne told MTV News. "He invited us down and we had a good listen, but there's no real collaboration or anything."
Bieber's manager Scooter Braun previously teased that a future collaboration between the two acts is "very possible".
One Direction will release their second studio album Take Me Home in the UK on November 12 and a day later in the US.
Watch the music video for Justin Bieber's latest single 'As Long As You Love Me' below: