The iconic Jamaican had to settle for bronze despite matching his season's best of 9.95 seconds, long-time rival Gatlin denying him a final slice of individual glory by three hundredths of a second.
Promising prospect Christian Coleman claimed the silver medal with a 9.94s run, but it was Bolt's name that was nevertheless chanted when the results were confirmed on the big screen at London Stadium.
The shock outcome represented the eight-time Olympic champion's first defeat in a major 100m final, bar his disqualification at Daegu 2011, but he matched Merlene Ottey's record 14-medal haul in the World Championships and can surpass it with a top-three finish in the 4x100m relay next weekend.
— IAAF (@iaaforg) August 5, 2017
Bolt has struggled with his start throughout his career and criticised the blocks in use at London 2017 after his heat. This time he paid the price for getting out slowly as he was dethroned by Gatlin, who was roundly booed throughout the competition having previously served two doping bans.
The American celebrated by putting a single finger up to his lips in response, having regained the world title 12 years after he first won it in Helsinki.
Gatlin, the oldest 100m winner in World Championships history at 35, burst out of the blocks and edged Coleman on the line to claim the win.
Bolt again raced through the field over the second 50m but his sluggishness off the blocks left a gap that was narrowly insurmountable. However, he was able to overtake compatriot Yohan Blake to go out with a medal.
Still, Bolt celebrated in front of the adoring crowd, taking a bow in recognition of the incredible support he has received.
Photo by Getty Images. Caption: Justin Gatlin celebrates his 100m final victory in London