The Bootylicious singer, whose husband Jay Z played a famous set at the 2008 festival, performed her solo repertoire, hits from her time as a member of Destiny's Child and an eclectic mix of cover versions.
The crowd helped the superstar through "Say my Name", "Survivor" and "Independent Women" along with her interpretations of the Eurythmics' "Sweet Dreams", Alanis Morisette's "You Oughta Know" and "Sex on Fire" by the Kings of Leon.
Addressing the massed ranks, the singer said: "I want you all to know right now you're witnessing my dream.
"I always wanted to be a rock star and tonight we are all rock stars," she added. "I still can't believe I'm performing at Glastonbury."
Earlier in the day, veteran singer-songwriter Paul Simon took to the main stage to perform hits "Diamonds on the soles of her shoes" and "You can call me Al", with a huge crowd seemingly unperturbed by his illness-affected voice.
British rapper turned soul crooner Plan B cemented his reputation as one of the country's hottest talents with an epic early evening set on the main Pyramid Stage.
"I came here in 2000, broke in, and watched Cypress Hill on the main stage," the singer told BBC. "To perform there and look from the other side was mental."
Despite a wet start to festivities on Thursday and Friday, the weather gods smiled on the 170,000 ticket holders with unbroken sunshine greeting artists performing at the farm in Somerset, south west England, on Saturday and Sunday.
Rockers Coldplay occupied the coveted Saturday night headline slot, while Irish megastars U2 kicked off proceedings at the world's largest performing arts and music festival with a two-hour set on Friday.
Crowd violence marred the band's debut Glastonbury performance as security guards stamped out a protest against the rockers' tax status.
As the band came on stage, campaign group Art Uncut inflated a 20-foot (six-metre) balloon emblazoned with the message "U Pay Your Tax 2."But as they tried to release it over the crowd, a team of security guards swooped into the crowd and tackled them to the ground before confiscating the balloon.
"Bono is well known for his anti-poverty campaigning but Art Uncut is accusing him of hypocrisy," campaigner Charlie Dewar said.
The group highlighted U2's decision to switch their tax affairs from Ireland to the Netherlands to pay less tax following a change in the law in 2006.
On a sombre note, the body of Christopher Shale, chairman of West Oxfordshire Conservative Association, was discovered Sunday morning in a portable toilet in the festival's VIP backstage area.
British Prime Minister David Cameron and his wife Samantha said they were "devastated" to hear of the death of their "great friend". (AFP)
