The Duke of Rothesay was joined on the Dumfries House Sport Relief Mile by the co-presenters of the BBC's One Show, Alex Jones and Matt Baker, as well as Scottish X-Factor finalist Nicholas McDonald.
About 100 young people from local schools and Sport Relief-funded projects set off on the run, which took them around the estate, near Cumnock in East Ayrshire.
Pipers played as 12-year-old Aidan Hessett, from Auchinleck Academy, crossed the finish line first.
Sports Relief activities - comprising activities such as running, swimming and cycling - will take place on the weekend of March 21 to 23. Events will take place at more than 1000 locations around the UK.
The money raised by the public will be spent by Comic Relief to help people in need in the UK and abroad.
Before sounding the klaxon to kick off the mile-long run, Prince Charles said: "I do hope all of you taking part will have some very good exercise and perhaps some considerable relief by the time you reach the end of the mile."
BBC presenter Ms Jones said after the run: "It was really quite refreshing to run this morning with all these young people, to do it in such nice surroundings, and to be the first to do the mile for Sport Relief 2014."
Mr Baker said: "What a way to start off, with His Royal Highness, with the hooting of the horn.
"He did warn us about the potholes and he was right, so he's obviously been out and set the course."
He added: "There's a great spirit here and everybody's in a fundraising mood. Let's hope it continues."
Singer Mr McDonald said: "It was a privilege to be invited, to do the mile and meet everyone, all the kids. It was such a good day, but I'm so unfit!
"I hope people get encouraged by this, though. I hope everybody gets their mile done, even if it's running to the shop and back, and you get a sweetie after."
Prince Charles led a consortium which bought Dumfries House and saved it for the nation in July 2007.
The house and estate have been developed as a visitor attraction, and the Prince visits several times a year.
The estate includes the Tamar Manoukian Outdoor Centre, which provides accommodation and team activities for schools and the uniformed youth services, the Belling Hospitality Training Centre, artists' studios and the Sawmill Traditional Skills Centre.