The group collaborated with the American country singer in 2003 to record 'Slow Dancing', which was written by Bono.
While a live version of the track, which featured on 'The Essential Willie Nelson' album, surfaced online, no official footage was previously available.
The five-minute video was posted on YouTube earlier this week, on U2's account as a tribute to Nelson on his 80th birthday, and it has already been viewed 10,000 times.
It is completely shot in black and white. The credit reads: "Happy birthday Willie! 80 and still a pilgrim. All the best from Bono, The Edge, Larry and Adam."
The band had previously collaborated fellow country legend Johnny Cash. U2 are currently working on their 13th studio album, provisionally entitled '10 Reasons to Exist'.
It has been reported that the band are hopeful of unveiling their latest offering to fans in September.