Native River has been ruled out of the Cheltenham Gold Cup with a suspensory tendon injury.
The 10-year-old won the race in 2018 and was aiming to become the first horse since Kauto Star in 2009 to regain the title.
Native River, trained by Colin Tizzard, eased to victory in the Denman Chase earlier in February.
Tizzard's son and assistant Joe said the injury meant the horse's "season is over".
Native River was also set to race in the Grand National but had not been confirmed for the race.