Stormy weather had threatened to delay the liftoff, but the spaceship was able to thunder into orbit at 11:29 a.m. ET, embarking on the 135th shuttle mission.
"Let's light this fire one more time," Commander Christopher Ferguson said just before taking flight.
The historic liftoff was 30 years and three months after the very first shuttle flight.
It will be at least three years - possibly five or more - before astronauts are launched again from U.S. soil, and so this final journey of the shuttle era packed in crowds and roused emotions on a scale not seen since the Apollo moon shots. NASA has set of long-term goal of flying to an asteroid and eventually Mars.
Atlantis is bound for the International Space Station, making one final supply run. The crew will deliver a year's worth of critical supplies and return with as much trash as possible.
From now on, private companies will take over the job of hauling cargo and crews to the space station.
Thousands of spectators were at the Kennedy Space Center to watch the launch. The spaceship is scheduled to come home on July 20 after 12 days in orbit.
Thousands of shuttle workers will be laid off within days of Atlantis' return.
Atlantis will then be put on display at the Kennedy Space Center. Discovery and Endeavour already are retired and being prepped for museums in suburban Washington and Los Angeles.
The Associated Press contributed to this report.