Stars were making last-minute preparations ahead of a trial run of the show when the fitness guru - whose real name is Derrick Evans - landed awkwardly after performing a straddle vault.
Programme medics rushed to his aid, giving him gas and air to ease the pain, and packed the injury - which includes tendon damage - with ice.
The 61-year-old - known for his years of getting the nation moving on GMTV - has now been ruled out of the BBC1 contest, which is due to launch in the coming weeks.
He had spent six weeks training for the programme and show bosses were working on a pilot version of the show to ensure ran smoothly before it goes on screen.
The BBC said: "Mr Motivator dislocated his knee when landing from a vault in rehearsal for Tumble yesterday. His patella tendon was ruptured and he will be undergoing surgery today. This means he will be unable to continue as a contestant on Tumble.
"The Tumble production team are extremely sad to see Mr Motivator leave the competition in this way and send him every good wish for a speedy and full recovery."
Programme chiefs are still considering whether they should replace him or continue the show with one fewer contestant.
Former Girls Aloud singer Sarah Harding, Dynasty actress Emma Samms and boxing champion Carl Froch are among the other stars who have signed up.
The Saturday night entertainment show will feature a range of disciplines and the injury is bound to unnerve the other contestants, who also include Ian "H" Watkins from pop group Steps and Loose Women panellist Andrea McLean.
Evans said: "I'm devastated not to be able to continue on this amazing experience I have had so far.
"A heartfelt thanks goes to all the team but especially my gymnastics partner Kerry and coach Alberto, who have been so supportive and brilliant to work with - I'm sorry our journey has been broken this way."
He added: "Good things sometimes fall apart but from it, always remember, we can only get stronger. Good luck to all cast and crew of Tumble."
The series is to be hosted by One Show presenter Alex Jones and judges will include Olympic gymnast Nadia Comaneci and the London 2012 medallist Louis Smith, who is already a seasoned veteran of celebrity shows after triumphing in Strictly Come Dancing.