Little Josh has become the second horse to die at Aintree ahead of the Grand National, after a shoulder fracture.
Having sustained the fatal break in a fall at the 15th fence in the Topham Chase, vets put down the Nigel Twiston-Davies-trained National Hunt racer.
The British Horseracing Authority confirmed Little Josh was "humanely put down".
Little Josh competing at last year's meeting at Aintree
Professor Chris Proudman, veterinary advisor to Aintree, said: "We are sad to confirm that is has been necessary to humanely put down Little Josh on welfare grounds, as a result of his fall at the 15th fence in the John Smith's Topham Chase.
"He received immediate veterinary attention for a broken shoulder, but this injury was not treatable and it was the necessary course of action."
All 28 other horses in the race returned safely.
Aintree said jockey Liam Treadwell received medical assessment on course and was taken to hospital for further assessment.
Little Josh had previously shown smart form over the big fences, finishing seventh when favourite for this race a year ago and winning the Grand Sefton Chase in December.
He fell on his next appearance at Cheltenham and was pulled up in the Denman Chase at Newbury.
Yesterday, Battlefront was pulled up during the fourth race by jockey Katie Walsh and later collapsed and died. It is thought Battlefront may have suffered a heart attack.
A further five horses fell in the race, although none was significantly injured, and Andrew Tyler, director of Animal Aid, said: "The Aintree authorities and the British Horse Racing Authority have been claiming that major new safety measures and efficiencies would eliminate much of the risk associated with racing on the Grand National course.
"But today's Fox Hunters' Chase, in which Battlefront lost his life, was stomach-wrenchingly chaotic from start to finish. Several horses fell or were pulled up, tired and potentially injured.
"It was both utterly depressing and served as confirmation that the Aintree authorities have got it badly wrong once again."