He developed a muscle pull and was unable to finish the 4×100 relay final for his country.
It was an agonizing end for the former world champion, who now formally retired today.
He was running the final leg of the World Championships 4×100 meters relay as Britain beat the United States for a shock gold medal.
Bolt, who had to settle for bronze in the individual 100 meters, had been hoping to sign off from the sport by leading Jamaica to a fifth successive relay gold but they were already struggling in third when he collected the baton.
As he tried to gain ground, Bolt pulled up and fell to the floor with what looked like a hamstring injury.
The United States, with individual gold and silver medalists Justin Gatlin and Christian Coleman running the second and fourth legs, had been expected to push the Jamaicans all the way.
Instead, a brilliantly executed race by Chijindu Ujah, Adam Gemili, Danny Talbot and Nethaneel Mitchell-Blake gave Britain gold in 37.47 seconds, the best time in the world this year.
The US took silver in 37.52 with Japan third in 38.04.