Best Mate's jockey Jim Culloty has stunned racing by retiring because of health reasons.
Culloty won the Gold Cup and the Grand National in 2002
The Irishman, who partnered the horse to three successive victories in the Cheltenham Gold Cup, also won the 2002 Grand National on Bindaree.
"I have been thinking about it for some time," he said. "Whenever I have had a fall lately I have been left feeling dazed, which I didn't in the old days.
"As much as I love riding, I have to put my health before my career."
Best Mate was set to bid for an historic fourth Gold Cup victory in March before a late withdrawal after bursting a blood vessel on the gallops.
Trainer Henrietta Knight will now seek a replacement for Culloty when Best Mate is aimed at wrestling back his crown from 2005 winner Kicking King.
She said of Culloty: "He is a very good friend of ours and we have had some wonderful times together, most notably when winning three Gold Cups."
"He had worries about the falls that he has had recently and he has got to put his health before everything else."
It had been confirmed in May that Culloty, 31, would not be stable jockey to Knight next season, although he was expected to keep the ride on Best Mate.
Best Mate's owner, Jim Lewis, said at the time: "As long as Jim is available to ride Best Mate and my other horses, he will always be there as number one choice."
Born: 18/12/73, Killarney
First winner: Karicleigh Boy, Exeter, 1994
Best Mate 2002, 03, 04
National: Bindaree 2002
Edredon Bleu 2003
Irish National: Timbera 2003
Culloty rode Best Mate on all three of his Cheltenham blue riband triumphs - in 2002, 2003 and 2004.
In 2002, he completed a famous double by winning the National at Aintree on Bindaree after being called up as a late replacement.
The following year, he won the Irish Grand National aboard Timbera.