Hayne shook his head, swore and went to the bench.
There were 70,499 fans in the stadium, about 15-million more across America watching the Monday night football telecast and many more in Australia.
It was a horror moment for Hayne.
But, there was a happy ending for Hayne and his 49ers with the team scoring a comprehensive 20-3 victory in their first game of the new season.
Hayne was livid after that first-quarter fumble and, with all of the hype he had received, he could have been ostracised by his teammates.
But his team leader, quarterback Colin Kaepernick, quickly came to his side and offered encouraging words.
Bruce Ellington, who Hayne beat to be the 49ers' first-choice punt returner, also came over.
49ers head coach Jim Tomsula and special teams coordinator Thomas McGaughey sent Hayne straight out for the next punt return, which he caught safely.
Ellington would be called on to catch punts later in the game, but that was because Hayne suddenly became the 49ers' second running-back option when veteran Reggie Bush suffered a game-ending leg injury.
49ers' running back star Carlos Hyde was sensational with 26 carries, 168 yards and two touchdowns to lead his team to the win.
Hayne came in to give Hyde chances to take rest breaks.
Hayne had 13 yards off four carries and a catch for seven yards.
"I still have a long way to go and learned a tough lesson tonight," said Hayne in the locker room, surrounded by reporters and rap music blaring in the background.
A little more than a year ago, he announced he was leaving Parramatta NRL club and Australia to pursue his NFL dream in the US.
Just over a month ago, he had his first helmet and pads tackle practice session.
On a windy night in front of millions, he completed his first fully fledged NFL game.
It wasn't until a fan tossed an Australian flag to him after the win and he wrapped it around his shoulders that the significance struck him.
"It was a bit emotional seeing that flag and it kind of brought back memories really of how far I've come and people supporting me," Hayne said.
The 49ers travel next weekend to play the Pittsburgh Steelers.
- AAP