Engineers had to scramble to diagnose a problem with a pressure valve on the first stage of the Delta 2 rocket in order to get the rocket off at 0218 GMT, 43 minutes late.
The $400 million mission finally got away in spectacular fashion, streaking across the night sky over Florida like a massive fireball, lighting up the beaches below.
The lift off from Cape Canaveral ended nearly two weeks of frustrating delays for the space agency which saw the launch being postponed five times.
When the six-wheeled Mars Expedition Rover "Opportunity" reaches the Red Planet, it will scour the surface for signs that could point toward ancient life.
Already speeding toward Mars is the first of the twin rovers, named "Spirit," which was launched on June 10. Both are expected to land on Mars, 9,600 km apart in January 2004.
NASA said that scientists do not expect the rovers to find life there, or even direct evidence of ancient life, but they hope to determine whether conditions were favourable on Mars three billion to four billion years ago for life to evolve.
But the twin Rovers face not only a long journey across more than 483 million km of interplanetary space, they must also land on Mars using parachutes to break their fall and airbags to cushion their landings.
Of nine spacecraft that have tried to land on Mars, only three have succeeded.
Two other missions, one European and the other Japanese, are already headed for Mars. Like the Rover twins, they are taking advantage of a very rare proximity between the two planets that has cut the travel time to seven months from the usual nine to ten months.