Eclair Surf has become the second equine fatality at the Grand National following Discorama on Saturday.
Trainer Emma Lavelle said the horse suffered a traumatic head injury during Aintree’s showpiece race.
It is the first time two horses have died in the race since 2012, after which safety changes were brought in.
It is also the fourth equine death across the three-day Aintree meet after the mare Elle Est Belle earlier on Saturday and Solwara One on Friday.
Eclair Surf, ridden by Tom Bellamy, won the Classic Chase earlier this year.
He was stabilised on the course following his fall and was treated by vets at Aintree before being transferred to Liverpool University.
His condition deteriorated on Sunday morning and was then put to sleep on welfare grounds.
“He was an incredibly special horse, loved by everyone and he will be missed terribly,” Lavelle said on Sunday.
Significant changes were made before the 2013 Grand National.
The core of the fences was softened, the distance reduced and new procedures were put in place for loose horses.
There have now been four equine fatalities from 356 runners in the nine Grand Nationals since 2012.
James Given, director of Equine Health and Welfare at the British Horseracing Authority, said safety in the sport was “an ever-evolving commitment”.
“Every incident this week will be reviewed, and this information will then build on the significant evidence and evaluation that took place as part of the 2011-12 review,” Given added.
“Should any further trends be identified that might be linked to increased risk then we are able to act on them.”
Noble Yeats won Saturday’s race in a thrilling finish.