Definitly Red won 15 of his 38 career starts, including the Many Clouds Chase at Aintree twice, the Charlie Hall Chase at Wetherby and the Cotswold Chase at Cheltenham.
The 12-year-old was running in the world's most famous steeplechase for a second time, after being badly hampered when still going well in the 2017 renewal.
Trainer Brian Ellison posted on his Facebook page: "The decision has been made to retire Definitly Red after his run in yesterday's Grand National. He pulled up two fences before the finish when getting tired, but delighted to report that he came home safe and sound.
"We, along with owners Phil and Julie Martin, have been honoured to have had such a wonderful horse. He has given us all so much pleasure and owes us nothing.
"We would like to thank everyone who has supported and followed 'Red' throughout his career."