Red Cadeaux, ridden by Gerald Mosse, finished second nearly five lengths clear of Ryan Moore on Planteur who, like last year, came home third in the world's richest horse race.
Animal Kingdom, the 2011 Kentucky Derby winner trained by Englishman Graham Motion, held close to the pace for most of the 10 furlong (2,000 metres) race, taking over the lead from top US mare Royal Delta in the final turn to give America victory.
"It's a tremendous horse. It's unbelievable," Rosario, who made his move 2-1/2 half furlongs out, said after the race on the synthetic track. "To win the World Cup - it's great."
"He broke well today and he put himself close to the pace and took himself where he wanted to be. In the turn for home he was like look around where is my competition so that was awesome," Rosario added after the race.
The five-year old chestnut, owned by Arrowfield Stud and Team Valor, ended a three-year drought for US-bred runners in the final race of the night at the opulent Meydan course.
"He reminded me very much of the (Kentucky) Derby today the way he ran, it was the same kind of run he made home he just seemed so confident," Motion said.
Red Cadeaux, trained by Ed Dunlop, was priced at 28-1 and Planteur, recently bought by a member of the Qatari royal family, was a 10-1 shot.
Asked about the prospects of running Animal Kingdom at Royal Ascot this year, John Messara, one of the owners, said: "There are risks and logistics involved."
"So we need to sit down (with a) cool mind tomorrow and have a bit of chat about it with (Team Valor CEO) Barry (Irwin) and Graham (Motion as to) where we go and what the risks and rewards are," he said.
Double Breeders' Cup Ladies Classic winner Royal Delta, 4-1 favourite and ridden by U. Hall of Fame jockey Mike Smith, faded in the home stretch to finish 10th after leading for much of the race.
Last year's Dubai World Cup winner Monterosso, owned by the Godolphin operation of Dubai ruler and United Arab Emirates Prime Minister Sheikh Mohammed bin Rashid al-Maktoum, was pulled out of the race earlier on Saturday.
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