The band had to cancel their scheduled appearance at one of the world's biggest outdoor music events last year when lead singer Bono injured his back.
U2 will play the Pyramid stage, which can be seen by tens of thousands of fans standing on a grassy slope, on Friday, June 24.
"After the disappointment of U2 being unable to play last year, we're now doubly excited at the prospect of the Friday night supergroup breaking the sound barrier," said Glastonbury organiser Michael Eavis.
U2 guitarist the Edge added: "It's more a way of life than a festival. I made it last year to the Friday show - got to play with Muse, got to hang out, ate a veggie burger and fries, and got to play on the Pyramid Stage.
"I have to say there is something really special and iconic about that stage. So we're all looking forward to coming back to pick up where I left off. And we're so excited to get to play in front of the world's greatest festival audience."
Eavis, an English dairy farmer now in his 70s, has developed Glastonbury from a small, free music festival into a major event in the pop calendar attracting some of the biggest acts and around 150,000 paying fans living on site in tents.
The Edge actually did play with Muse last year as they teamed up for a version of Where the Streets Have No Name.