The British rockers - consisting of Matt Bellamy, Chris Wolstenholme and Dominic Howard - will play the Friday night of the festival in Somerset, England, later this year.
Muse - who announced the news on their Twitter page - will now become the first band to headline all three nights of the prestigious music festival, as they previously topped the bill on Sunday in 2004 and Saturday in 2010.
They join Coldplay who will perform on the main stage on closing night, making them the first act to top the bill at the festival four times.
And Emily Eavis - who organises the event with her father Michael Eavis - recently revealed that all three headliners are British.
She said: "We've got three brilliant British bands headlining the festival."
While she did not confirm Saturday's act, Emily revealed she is extremely excited that Chris Martin and Coldplay are returning to Worthy Farm and admits it feels like a homecoming to have them play again.
She told BANG Showbiz: "Glastonbury is like their homecoming they've been all over the world they've the Super Bowl and now they're coming back to the West Country.
"It's mean a lot for us to have them back, we've always had immense pride in seeing them go off around the world and become so big. They started at Glastonbury in the new band tent in 1999 and then they headlined the Pyramid Stage two years later off two albums which is pretty impressive. It's been an incredible journey and for them to come back here for a fourth time I think is amazing."