Simona Halep said she was "really down and upset" at not being able to defend her ladies singles title at Wimbledon due to a calf injury.
The 29-year-old Romanian had been battling to be fit since withdrawing from her second round match at the Italian Open in mid-May.
"It is with great sadness that I'm announcing my withdrawal from The Championships as my calf injury has not fully recovered," she said.
Halep's withdrawal came minutes before the draw for the championships which get underway on Monday.
Halep secured her second Grand Slam singles crown beating Serena Williams in 2019 -- due to the coronavirus pandemic the Grand Slam event was cancelled last year.
She joins four-time Grand Slam champion Naomi Osaka in withdrawing.
With doubts also over the fitness of world number one Ashleigh Barty, the cards could be falling right for 39-year-old Williams as she seeks a record-equalling 24th singles Grand Slam title.
Halep said missing the French Open and now Wimbledon had been trying for her mentally.
"I gave it everything I had in order to be ready to play Wimbledon and after having such special memories from two years ago, I was excited and honoured to step back on these beautiful courts as defending champion," she said.
"Unfortunately, my body didn't cooperate and I'll have to save that feeling for next year.
"I can honestly say that I'm really down and upset about having to take this decision.
"This period has been difficult but to miss the last two majors has made it even more challenging mentally and physically."