Minella Reception has died after a fall at Becher's Brook in Friday's Topham Chase at Aintree.
The 10-year-old, trained by Nigel Twiston-Davies, was treated on the course but then put down after assessment back at the stables.
Gullinbursti had earlier been put down after falling in the same incident.
It takes the number of horse deaths to four at this year's Grand National meeting after Clonbanan Lad and Marasonnien collapsed on Thursday.
Neither fell but they died after being pulled up in the Fox Hunters' Chase which, like the Topham Chase, is staged over the same course as Saturday's Grand National.
Thirteen horses have died at the Aintree meeting in the past five years.
The British Horseracing Authority said there was one fatality from 661 runners at the previous two National meetings.
Since modifications to the fences and other changes three years ago, there have been no fatalities in the Grand National itself.
"While you can't remove all risk from any sport, we acted on evidence to make significant changes here at Aintree, including to the cores of every fence on the Grand National course," said John Baker, the north-west regional director for Aintree's owners Jockey Club Racecourses.
"We've seen hundreds of horses compete safely since over the last few years."
Speaking on the opening day of the meeting, Eduardo Goncalves, chief executive of campaign group League Against Cruel Sports, said: "We've seen some improvements in some places, but not enough in many."
The League Against Cruel Sports recommends removing the Becher's Brook fence where Gullinbursti and Minella Reception were fatally injured and reducing the field of any race to a maximum of 30 runners.
Marasonnien was ridden by amateur jockey Patrick Mullins - son of Irish champion trainer Willie Mullins - while Clonbanan Lad, who won last time out at Fakenham, was from Louise Allan's yard.