Heffernan earned his first victory at the meeting ahead of Flintshire, with another O'Brien runner, Found, third.
Meanwhile, Arrogate surged past California Chrome in the closing yards to win the Classic.
Frankie Dettori rode Queen's Trust to victory for British trainer Sir Michael Stoute in the Filly & Mare Turf.
Highland Reel gave O'Brien his 22nd Group One flat race win of 2016 - three behind the all-time record of the late American trainer Bobby Frankel.
"Seamie has given this horse a star ride," said the trainer.
The winner will continue racing next year, while Prix de l'Arc de Triomphe winner Found - who was ridden by Ryan Moore - will be retired for a breeding career.
Arrogate, ridden by Mike Smith, stole the limelight from favourite California Chrome with a storming finish to win the showpiece $6m (£4.8m) Classic by half a length.
It was a third consecutive Classic win for trainer Bob Baffert, following triumphs with American Pharoah and Bayern.
"It's incredible that I've been so lucky, someone up there is really looking down on me," Baffert said.
Dettori treated a crowd of more than 70,000 to a trademark flying dismount from Queen's Trust after pipping home favourite Lady Eli on the line.
"Sir Michael is a genius and he was very sweet on this filly," Dettori said after his 12th win at the meeting.
BBC racing correspondent Cornelius Lysaght
Seamie Heffernan 'stole' victory on Highland Reel - riding the son of stallion Galileo into a considerable advantage with still a third of the mile and a half-long race to go.
It was a bold move because a jockey can look like a right clown if he or she grabs a big lead and gets caught close to the finish - and the other riders probably thought he would run out of puff in front.
But Heffernan, long a big part of the Aidan O'Brien/Coolmore operation in Tipperary, knew what he was doing, and had the last laugh.
Highland Reel won the Breeders' Cup Turf at Santa Anita for Irish trainer Aidan O'Brien after a brilliant front-running ride from Seamie Heffernan.