Barney Roy upset Churchill by winning the St James's Palace Stakes for trainer Richard Hannon and jockey James Doyle on day one at Royal Ascot.
The 5-2 winner provided Godolphin with their second victory of three on the day when it came home ahead of Lancaster Bomber in a course record one minute and 37.22 seconds, with Churchill having to settle for fourth position.
Barney Roy finished as runner-up to favourite Churchill in the 2,000 Guineas at Newmarket last month, but the colt came out on top this time around.
Godolphin's first triumph in their Group One double on Tuesday came courtesy of Ribchester - 11-10 favourite - in the Queen Anne Stakes.
The King's Stand Stakes was won by Lady Aurelia, ridden by John Velazquez in place of the injured Frankie Dettori, who famously won all seven races on a single day at Ascot back in 1996.
Thomas Hobson won the Ascot Stakes, with Rajasinghe coming home first in the Coventry Stakes before Sound And Silence competed Godolphin's treble in the Windsor Castle Stakes.