Over the course of the weekend, more than 2,000 acts will play on the festival's stages - but all eyes will be on the main headliners: Radiohead, Foo Fighters and Ed Sheeran.
All three spoke to Radio 1's Annie Mac this week, discussing their hopes and fears for the festival.
Here's what they had to say, and a look at their history at Glastonbury.
Radiohead are headlining Glastonbury for the third time, 20 years after they first topped the bill.
That show, played just two weeks after the release of OK Computer, was named the best gig of all time by Q Magazine but Thom Yorke recently revealed he nearly walked off stage in frustration after the band's monitors blew up, leaving them unable to hear each other.
"I just went over to Ed [O'Brien, guitarist] and said, 'I'm off mate, see you later,'" he told BBC 6 Music. "He turned around and went, 'If you do, you'll probably live the rest of your life regretting it.' I went, 'Good point.'"
In the intervening years, Radiohead have done more than any other band to push the boundaries of rock music, stretching dark, brooding electronics over Yorke's keening vocals. Their most recent album, A Moon Shaped Pool, rekindled their relationship with melody, and is bound to form the core of their set on Friday night.
Annie Mac: How are you all feeling about this?
Ed O'Brien: To be honest, a little nervous. A few days ago we were doing the setlist for a show in Denmark and we all fessed up: We had the Glastonbury tingles. You know, that anticipation and slight nerves. It means a lot. It's a huge one.
What does it feel like to stand on that stage and look out to 100,000 people?
The first time we played it in '97, it felt like we were looking out upon this scene of devastation. The rain was horrendous.
But the interesting thing is, when it's really right, it doesn't feel like there's a divide. There's a feeling of the band and the audience experiencing this thing together.
Can you tell us anything at all about what you'll be doing on Friday night?
There won't be any sort of guest appearances! I thought that Coldplay with Barry Gibb last year was brilliant [but] we're not that kind of band.
What advice would you give to people who are virgin Glastonbury headliners?
It's all about humility. For me, the bands who don't do it on that stage - or anywhere in Glastonbury - are the ones who turn up with their shades on, and it's all about them.
You've got to remember, you're just closing the night. You're not headlining, you're one part of this huge, great, amazing beautiful festival. You're providing maybe two hours of soundtrack to people's enjoyment and experience at that moment.
You've got to leave your ego and shades at the gate.
Foo Fighters were all set to headline Glastonbury two years ago when Dave Grohl fell off stage and broke his leg in Gothenburg, Sweden. With two weeks' notice, Florence + The Machine were drafted in to take the band's slot, and Grohl spent the rest of the year recuperating (and performing from a specially-constructed throne). Now, he's back on two feet, and ready to rock.
Powered by a seemingly endless supply of both hit singles and rock star charisma, the band are perfect headliner material.
Fist-pumping anthems like The Pretender, Best of You and Times Like These are likely to be joined by new single Run, but hopefully the band will avoid the temptation to break out brand new tracks from the forthcoming album Concrete & Gold - "a record that sounds like Motorhead doing Sgt Pepper's," according to Grohl.
Expect the mosh pit to be full for this one.
Annie Mac: Would it be right to say this show will be a pinnacle in your career?
Dave Grohl: We last played Glastonbury in 1998 and we were in our infancy then. We were halfway down the bill, it was pouring with rain and I think there was some sort of Euro cup final thing happening, so about 90% of the audience disappeared once we hit the stage.
We were supposed to be back a couple of years ago and unfortunately I had to call in sick. So this is a big make-up date for me. We get to headline Glastonbury but also I get to do it standing on two legs. It means a lot to me, personally. It's part of my recovery in a weird way.
You've had two extra years of a build-up to this headlining slot. Are you feeling like it's going to be better than it was then?
Now there's more purpose to us playing the gig.
You know, Florence + The Machine took our place as headliners and I saw footage of them playing one of our songs, Times Like These, and I got really emotional and thought, "What a beautiful gesture". But [it was] also a beautiful moment that connected the band and the audience, so I can't wait to play that song.
I've been thinking about this for two years. Playing a song like Times Like These in front of that audience for the first time, standing up on two legs, is huge. It's a big deal, personally.
In just six short years, Ed Sheeran has graduated from Glastonbury's tiny Croissant Neuf stage to the top of the bill.
He proved his ability to get people on his side in 2014, when he inherited Dolly Parton's record-breaking Pyramid Stage audience, and got them all waving their shirts in the air during Sing.
Amazingly, he does all of this by himself. No band, no backing singers, no pyrotechnics. It's just Ed, slapping his hand against his acoustic guitar and layering up harmonies on a loop pedal, like a digital one man band.
Music snobs might turn their noses up at his more saccharine songs, but the Sunday night slot is traditionally reserved for an artist who can bring the crowd together for an undemanding sing-song. And in that respect, Ed fits the bill perfectly.
Annie Mac: How are you feeling about this weekend?
Ed Sheeran: Really, really, really excited. I'm actually more excited about this than I was about playing Wembley [Stadium] - because when you're playing your own shows, you're not really winning anyone over. They've all parted with cash to buy a ticket.
Knowing that there are people in the audience that possibly don't like my music at all... that excites me.
What's the plan for the set?
I'm doing it all by myself. I think the key to any festival set is just play songs that people know. I'm not going to be like, "here's a new song that I wrote last week". It's all going to be songs people have heard. Some [of the] songs I don't even play in my set anymore because they've been over done, but I'm remembering that this is the first time that a lot of people are going to see me.
What about getting some special guests out... like Stormzy?
Stormzy is playing on Friday, so I don't think he's going to be around. But Beoga - who are the Irish band that play on Galway Girl - are there on Sunday, so I might get them up and have a bit of a jam.
Will you be at the festival all weekend?
I don't like big crowds of people, ironically! So I'm not a big festival-goer.
I'm actually taking the opportunity to be at home for two days and then I'm going to go in on Sunday. And I've got every single member of my family coming so it's going to be like a wedding reception afterwards.
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After two days of build-up, Glastonbury kicks off in earnest on Friday when the Manchester Camerata open the Pyramid Stage, playing orchestrally-enhanced versions of club classics.