Liftoff took place Monday at 5:56 a.m. PT from the Kennedy Space Center in Cape Canaveral, Fla.
Up to 45,000 guests jammed into NASA's launch site. The shuttle quickly disappeared into the clouds within seconds of liftoff.
Just before launching, Commander Mark Kelly made some patriotic remarks: "It's in the DNA of our great country to reach for the stars and explore. We must not stop. To all the millions watching today, including our spouses, children, family and friends, we thank you for your support."
Endeavour is bound for the International Space Station one last time before heading to retirement at a Los Angeles museum. The shuttle's experienced, all-male crew of six will deliver and install a $2-billion particle physics experiment during the 16-day flight, as well as spare station parts.
Kelly is married to wounded Ariz. Congresswoman Gabrielle Giffords. Giffords, who was injured in January during a shooting at a political event in Tucson, was among the spectators at the Kennedy Space Center. The astronauts wore blue bracelets to celebrate Giffords' life.
Endeavour was previously scheduled for takeoff April 29, but an electrical problem delayed the launch.
This is NASA's next-to-last shuttle flight. Atlantis is scheduled to lift off this summer, bringing the 30-year shuttle program to an end.
The Associated Press contributed to this report.