Hill Sixteen died after falling at the first fence of the delayed 2023 Grand National at Aintree, the third horse fatality at the three-day meeting.
The 10-year-old, ridden by Ryan Mania, suffered an "unrecoverable injury", according to the Jockey Club. Horses had to bypass the fence on the second circuit while Hill Sixteen was tended to.
Two other horses in the race - Recite A Prayer and Cape Gentleman - were treated on course and taken away by horse ambulance for further assessment.
A total of 118 people were arrested after protesters - demonstrating against the staging of the race - delayed the start of the 175th running of the famous steeplechase by gaining access onto the racecourse in Merseyside.
Earlier in the day Dark Raven, a six-year-old horse, was put down after a fall in the Turners Mersey Novices' Hurdle, with jockey Paul Townend on board.
And on the first day of the meeting on Thursday, Envoye Special died over the Grand National fences in the Foxhunters' Chase.
Four horses died at the Aintree meeting last year, including two that were injured in the Grand National, which is the climax of the annual race meeting.
There have been five fatalities from 395 runners in the 10 Grand Nationals raced since safety changes were introduced in 2012.
Dickon White, who runs Aintree as North West regional director for the Jockey Club, said: "Hill Sixteen was immediately attended by expert veterinary professionals during the Grand National, but sadly sustained a fatal injury.
"Our heartfelt condolences are with his connections."
Animal rights charity Peta UK said the Grand National is one of the "most hazardous" races in the world and called on the public to urge sponsors to withdraw financial support for the event.
Campaign group Animal Aid said jump racing should be banned, adding: "Another innocent horse has their life taken from them in the name of entertainment and gambling."
Hill Sixteen's owner Jimmy Fyffe told Racing TV: "I am absolutely gutted, especially for [trainer Sandy Thomson] and all his team and all the family have come down to see him. It was heart-breaking.
"He could be in a field running about and you can lose a horse. The horses get looked after so well by all trainers. I've been in all the stables that I've got horses at and they are looked after like kings.
"They have a great life, they love running so I've not got any qualms with staying in this game."