The Take That star sings George Harrison's track, which featured on the band's 1969 Abbey Road album, as well as making a campfire cameo during the 90-second commercial.
The latest advert marks the return of Myleene Klass and Dannii Minogue, alongside Twiggy, Jamie Redknapp, Noemie Lenoir and Lisa Snowdon.
Barlow said of his involvement: "It's great to be back with M&S and it's real a privilege to cover such an iconic track. You can't better perfection but I hope we've given it a modern twist that will capture the mood of the nation and provide the perfect anthem for summer 2012."
The group are seen having fun in a park, while Minogue takes on Klass in an egg and spoon race.
Steve Sharp, executive director of marketing for the company, commented: "It's going to be the summer of a lifetime for our customers and we want to do something extra special to mark this celebratory year.
"Just like the Royal Family and our sporting heroes, M&S triggers a unique reaction in the hearts and minds of the British public and this campaign really encapsulates that sense of fondness and nostalgia."
The advert will receive its television debut during Emmerdale on ITV1 tonight.
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Watch Minogue and Klass talk about filming together below: