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."