Stampede spokeswoman Bonni Clark says in a news release that the 12-year-old thoroughbred was being ridden by an outrider during the fourth heat of the chuckwagon races Friday evening.
Clark says the horse and rider were returning to the infield when the animal collapsed.
She says a veterinarian was on the scene and determined that the horse died immediately.
Clark says the horse wasn't involved in a collision and there was no obvious sign of injury.
Earlier this week, a steer at the Stampede had to be euthanized due to a severe neck injury it suffered during a rodeo event.
"We acknowledge that the death of an equine athlete in a public event may be upsetting for some people," Clark said in the news release.
Outriders are part of the chuckwagon team and follow the wagon during the race to the finish line.
The Stampede said the horse that died, named Hughey, was part of Doug Irvine's team and had been racing with his team for five years.
One of Irvine's horses died of a heart attack while doing training exercises at the Calgary Stampede in 2010.
Six horses died at the Stampede that year. In 2011, the Stampede adopted new rules for rodeo events and chuckwagon races to try to provide more protection for both human and animal participants.
The death on Friday prompted the Vancouver Humane Society to reiterate its call for the Stampede to suspend the chuckwagon race. It said it wants "an independent panel of experts review the event to determine if anything can be done to make it safe."
"It is getting harder for the public to believe that these deaths are just coincidence or bad luck. There is something inherently unsafe about the race. Nothing the Stampede has done has stopped horses dying," the society stated in the release.
Clark said Hughey had been cleared to compete prior to the race as part of the Stampede's "Fitness to Compete" program and all signs were normal.
"Our thoughts are with Doug and his outfit on the loss of Hughey," Clark said.
The steer that died earlier in the week could not get up after having its head and neck twisted to force it to the ground as part of the competition. Three veterinarians in the arena made the decision to euthanize the animal.