The American, favourite to win the title after victories in his last three tournaments, slipped on stairs in his rental house on Wednesday.
The guessing game continued right up until the moment Johnson appeared on the first tee with playing competitors Bubba Watson and Jimmy Walker then walked away without taking a shot.
Johnson arrived at Augusta National around mid-day and warmed up under the watchful eye of swing coach Butch Harmon. The situation appeared dire as Johnson took a few cautious swings.
The big-hitting American was in clear discomfort unable to bend over leaving his caddie to tee up his ball. With his 2:03 tee time approaching Johnson was in deep discussion with his team on the practise range and told a television reporter he was going to give it a try.
A grim-faced Johnson made his way to the first tee but after a few swings turned and walked to the clubhouse. It was later announced he was withdrawing from the tournament.
I just don't feel like there is any chance of me competing. It hurts. I was doing everything I could to try to compete. I was up all night trying to get ready for today. I can't make my normal swing and I just don't feel like there is any chance I can compete. Obviously I want to play more than anything. I just can't swing the club. It sucks. I am playing maybe best golf of my career and this is one of my favourite tournaments. It sucks, it sucks really bad.
The last number one to miss one of golf's majors due to injury was Rory McIlroy at the 2015 British Open and before that Tiger Woods at the 2014 Masters.
Jordan Spieth was cruising along on his revenge tour of Augusta National when a late quadruple-bogey undid what had otherwise been a steady start to the U.S. Masters on Thursday for the former champion.
Shortly after safely navigating the par-three 12th - site of the infamous collapse that cost Spieth the tournament last year - the American world number six suddenly blew up at the par-five-15th after a poor club selection. Spieth found water with his third shot, and then sailed his next one over the green before chipping on and three-putting for a nine after going birdie-bogey over his previous two holes.
"I didn't take my medicine and hit it with a club that takes the spin off," Spieth told reporters about his shot that landed on the green before sucking back down a slope and into the water. "Instead, I was stuck in the '15's a birdie hole' mentality and it kind of bit me a little bit."
Spieth birdied the next hole en route to a three-over-par 75, six shots back of clubhouse leader William McGirt in the opening round of the year's first major.
Read the original article on Eurosport: World number one Johnson pulls out of US Masters on first tee, Spieth struggles