The group turned down offers to play the country's most prestigious rock music event for years, but embraced the down-to-earth spirit of the festival Saturday.
The Stones opened with "Jumpin' Jack Flash" and gave fans a fistful of classic hits including "It's Only Rock 'n' Roll (But I Like It), "Paint It Black," "Wild Horses" and "Gimme Shelter."
During the group's performance, Mick Jagger thanked fans who had followed the band for five decades, and told newcomers, "do come again," before giving the crowd what it had been waiting for - an encore of "Satisfaction."
The Stones recently wrapped up the North American leg of their "50 and Counting" tour. They have two concerts scheduled for next month in London's Hyde Park.
During a BBC radio interview Saturday, Jagger gave no clue about whether the band he started with Keith Richards in 1962 will ever call it quits. Jagger said, "I've no idea," before telling an interviewer that he'd probably continue as long as he was wanted.
Guitarist Richards said the band was "destined to play Glastonbury."
"I look upon it as a culmination of our British heritage really," he said. "It had to be done."
The Glastonbury Festival, which was founded my Michael Eavis in 1970, is held on a farm near Pilton in southwest England.
Other performers on Saturday included Elvis Costello and Primal Scream. The three-day festival wraps up Sunday with a headlining set from Mumford & Sons.
The Associated Press contributed to this report.