Top hats were thrown skywards as Estimate, ridden by Ryan Moore, fought off Simenon in the closing stages of the 2 1/2-mile race. A rapturous reception from a crowd of 75,000 greeted Estimate into the winner's circle.
After the race, television replays of the Queen watching the closing stages from the royal box illustrated just how much victory meant to her. Features contorted with tension gradually gave way to the broadest of smiles as Estimate galloped to victory.
The same sense of elation was evident when Moore, who rode an exemplary race, later relayed how events on the track unfolded. "To do it for the Queen is fantastic," the jockey said. "To win in her colours is exceptional."
Even Moore's usually inscrutable features subsided to a broad grin as the Queen celebrated victory together with her racing and bloodstock advisor, John Warren, and Estimate's trainer, Sir Michael Stoute.
"It's a special thrill to win this race for the Queen," Stoute said.
"It will have given her enormous pleasure. Never mind being the Queen, she loves her racing."
The Queen usually presents the Gold Cup trophy to winning connections but this time the Duke of York stepped forward to give his mother a prize she has never won, despite 21 previous winners at the royal meeting. Her first, Choir Boy in the Royal Hunt Cup, was gained 60 years ago, the year of her coronation.
Estimate started 7/2 favourite for the Gold Cup and Moore played to the filly's strengths by keeping her close to the early pace. Once into the home straight Moore asked Estimate for her effort and she responded with gusto to resist Simenon by a neck.
Simenon's trainer, Willie Mullins, amplified the mood when he said: "I wasn't disappointed to be second - maybe for the first time in my life. It has been a fantastic day, and a fantastic result for racing."
Ladies' Day at Ascot was already bathed in emotion after Lady Cecil, wife of Sir Henry, who died last week, sent out Riposte to win the Ribblesdale Stakes 40 minutes before Estimate's victory. Sir Henry was an icon of the royal meeting, where he had saddled 75 winners over four decades.
Jockey Tom Queally and Lady Cecil, who took over training responsibilities on Sir Henry's death, found the first two days of the meeting a difficult experience. "It has been a tough week," Queally said. "A lot of people close to the boss are struggling emotionally and this means an awful lot to everyone at Warren Place (where Cecil trained in Newmarket)."
Generous applause rang out when Lady Cecil went to greet Riposte. "The reception was for Henry; he would have loved it," Lady Cecil said. "He had been planning for Royal Ascot since last year and we are just carrying on what he wanted."
In normal circumstances No Nay Never would have written the day's headline after the colt, trained in America by Wesley Ward, ran away with the Norfolk Stakes.
Despite being slowly away, No Nay Never established a clear advantage after a furlong before Joel Rosario settled him into a gentler rhythm.
It was a beautifully judged ride from a jockey who wanted to save something for the uphill finish.
No Nay Never also broke the two-year-old track record. When Ward was informed of the fact, he replied: "That's what we expected of him."