Britain's David Millar was the winner of the stage, a 10.3-mile sprint. He was timed in 19 minutes, 3 seconds, just 2 second faster than the American in a race against the clock around a futuristic theme park in western France.
Armstrong temporarily relinquished his yellow jersey, but finished well ahead of Germany's Jan Ullrich, Switzerland's Alex Zulle and Italy's Marco Pantani, all considered possible threats to his title.
France's Laurent Jalabert finished third, 13 seconds behind the leader, while Ullrich, the 1997 champion, was another second back.
Zulle, second overall last year, finished sixth, 20 seconds behind the leader. Pantani, the 1998 champion, was well back in 136th place, 2:36 behind.
It was a stunning victory for Millar, the only Briton in the race. He's a time-trial specialist, but was not expected to win in such a star-studded field.
"I never really thought about the possibility of getting yellow," said Millar, who planned to sleep Saturday night in his jersey. "To beat someone like Lance surprised me greatly.
"I didn't want to believe it until I saw him crossing the line. Beating him in one of his specialties was very moving."
Armstrong, as defending champion, was last to race and knew exactly what he had to do. At the first time check, he had the best time, but just failed to beat Millar in the end.
It was at the same venue last year _ Futuroscope Park _ that Armstrong, on the Tour's next-to-last stage, won a time trial to virtually assure his overall victory, just three years after being diagnosed with testicular cancer.
The cancer had spread to his brain and lungs, and doctors gave him only a 50-percent chance of survival. After surgery and intensive chemotherapy, he returned to cycling.
His victory by more than seven minutes last year in cycling's showcase event, confounded doctors, rivals and teammates. On his way to victory, he won all three time trials.
This year, Armstrong knew another win would leave his rivals _ some of whom branded his 1999 victory a fluke _ psychologically scarred before the real road racing begins.
But competition is stiffer, with the return of Italian Marco Pantani and Ullrich, both past Tour winners.
Road racing begins on Sunday with a 120-mile trek from Futuroscope to Loudun in northwest France. The Tour ends July 23 in Paris.
Tour officials were hoping that year's race would help repair cycling's battered image following the 1998 doping debacle when teams were kicked out after admitting to widespread drug abuse and police raided hotels searching for banned substances.
But three of the 180 riders were expelled on Saturday, just hours before the first stage was to begin. Ousted were Russian Sergei Ivanov, Italian Rossano Brasi and Slovenian Andrej Hauptman.
All failed a mandatory blood test aimed at detecting the use of erythropoietin, or EPO, a synthetic hormone that stimulates the production of oxygen-carrying red blood cells.
Riders who are found to have a red blood cell count above 50 percent are given a compulsory 15-day ban on health grounds.