Three-year-old colt Educator was the first horse to carry the royal purple, red and gold colours since Britain's longest-serving monarch died on September 8.
But there was no fairytale victory for Educator, bred by the Queen, in a handicap at Salisbury.
The 11-10 favourite, ridden by Tom Marquand and trained by William Haggas, finished several lengths behind 7-4 winner Okeechobee.
"The plan was to win and it didn't go to plan - that's horse racing," Marquand told the BBC.
"It's a new era and while exciting, it is sad. All I have ever known, like most people, is just the Queen and it's passed on to the King. It's fantastic in continuing her legacy because she put so much hard work and passion into it."
Maureen Haggas, the trainer's wife, said: "He (Educator) ran all right, but he's been a long time off the track and he's a big lad who is quite full of himself and he needs racing, basically."