Auguste Rodin's victory -- his sixth Group One success -- completed a double for O'Brien and jockey Ryan Moore on the day.
Illinois won the marathon Group Two Queen's Vase raced in blazing sunshine and in front of packed stands filled with many well known faces including former England football manager Roy Hodgson.
Auguste Rodin had come into the race on the back of two defeats this season but he looked much more like the horse that won last year's Epsom and Irish Derbies.
Two French challengers came at him in the final furlong but Moore found an extra gear and in the end he held them easily enough.
For Moore it was an 81st Royal Ascot winner -- drawing him level with Frankie Dettori, who is over from the United States for his daughter Ella's wedding and not riding at this year's meeting.
Moore paid tribute to O'Brien saying he is "very humble but a unique and special trainer."
O'Brien backed that up after the race blaming himself for the "blips" on Auguste Rodin's record.
"I was giving the wrong instructions, it was my fault but we have changed the strategy and we are now getting it right," said the 54-year-old Irishman.
"What makes him so good is he has a personality, and horses need personality to succeed.
"He is a very special horse and Ryan gave him a very special ride.
"He can go on and do anything really."'Big engine'
Earlier Moore had also been at the height of his powers as he steered favourite Illinois across the line ahead of stablemate Highbury, whilst another Irish runner Birdman was third.
Moore, 40, said the winner was far from the finished article although he had the potential to go on and win England's oldest classic the St Leger in September.
"He is still doing a few things wrong but thankfully he has a big engine," said Moore.
"He will get a bit more professional and be better for it."
Three-time British champion jockey Oisin Murphy also recorded a double, and three for the meeting having struck with Australian raider Afoora on Tuesday.
The 28-year-old Irishman produced a superb ride on Running Lion in the Duke of Cambridge Stakes, leading his rivals a merry dance from start to finish.
He added the Royal Hunt Cup to his laurels later on Wednesday, riding Wild Tiger he managed to repel a fast-finishing Sonny Liston.
"Saeed (bin Suroor, the trainer) provided me with my first Royal Ascot winner (Benbatl) and he's given me another one today," said Murphy.
"He's been integral in my career. I'm very grateful to him."
Trainer Karl Burke landed his second Queen Mary Stakes in three years in the opener.
James Doyle just held on on 22/1 chance Leovanni ahead of the fast-finishing Mighty Eriu for the 36-year-old's 19th Royal Ascot triumph.
It was a 1-2 for Qatar as the winner is owned by the Emir, Sheikh Tamim bin Hamad Al Thani whilst the second -- a 50/1 outsider -- is owned by Qatar Racing.
For Burke it was a welcome pick me up.
His hotly fancied runner Andesite on Tuesday had to be withdrawn from the Coventry Stakes after what he called a "freak accident" in his stable.
"Yesterday did not go to plan, but yesterday is yesterday," said Burke, welcoming home his sixth Royal Ascot winner
"This filly is uncomplicated and the win bodes well for the future."
