Envoye Special suffered a fatal injury in the Foxhunters’ Chase on day one of the Grand National meeting at Aintree.
The Keiran Burke-trained nine-year-old fell at a fence when running loose, having unseated rider James King.
Famous Clermont won the race, which is for amateur jockeys and takes place over the National fences.
Four horses died at the Aintree meeting last year, including two injured in the Grand National, which is the climax of the annual three-day meeting.
There have been four fatalities from 356 runners in the nine Grand Nationals raced since safety changes were introduced in 2012.
A British Horseracing Authority spokesperson said: “Our thoughts are with everyone connected to Envoye Special.
“No one will be more affected by this news than the trainer, owner and stable staff who have provided the horse with first-class care throughout his life.
“Following his fall he was attended immediately by one of the 13 veterinary surgeons on site, but the nature of the injury meant that, sadly, the horse had to be humanely put down.”
Animal rights activists have threatened to sabotage Saturday’s National and police say they will deal “robustly” with any disruption.
