Its thunderous rise into a bright morning sky over the Kennedy Space Center in Florida was watched by hundreds of thousands of spectators.
Among them was Endeavour commander Mark Kelly's wife, Gabrielle Giffords, the Arizona politician shot in the head by a gunman in January.
Doctors were satisfied enough with her progress to allow her to travel to Kennedy to see her husband's departure.
"This mission represents the power of teamwork, commitment and exploration," the commander said in a radio call to launch controllers moments before lift-off. "It is in the DNA of our great country to reach for the stars and explore. We must not stop."
Endeavour cleared the pad just after 0856 local time (1256 GMT; 1356 BST).
Kelly and his crew are on a path to rendezvous with the International Space Station (ISS) on Wednesday.
They will deliver a $2bn particle physics experiment, known as the Alpha Magnetic Spectrometer (AMS), and a tray of critical spare parts to the orbiting platform.
On Endeavour's return, the only active ship left in the US space agency's (Nasa) shuttle fleet will be Atlantis.
It should undertake its final mission sometime in in July.
On completion of the 30-year shuttle programme, America will use Russian Soyuz capsules to fly its astronauts to the ISS.
A number of US national commercial carriers should are expected to enter into service around the middle of the decade.
Nasa will then buy seats in these astronaut "taxis". The arrangement will represent a major change in the way the agency does business. No longer will it own the vehicles it uses to go to low-Earth orbit.
This servicing plan should free up resources for Nasa to build an astronaut Space Launch System capable of travelling beyond the station, to the Moon, asteroids and to Mars.
Crews move the primary payload for space shuttle Endeavour's STS-134 mission into the Payload Changeout Room on Launch Pad 39A at NASA's Kennedy Space Center in Florida.