Launch from Cape Canaveral (KSC); HST-SM-04; landing on the Edwards AFB.

STS-125 was the fifth and final space shuttle servicing mission to the Hubble Space Telescope (HST). Atlantis carried two new instruments to the Hubble Space Telescope, the Cosmic Origins Spectrograph and the Wide Field Camera 3.

Megan McArthur successfully grappled Hubble at 17:14 UTC, and at 18:12 UTC the telescope was safely berthed in the payload bay of Atlantis. Later in the day, John Grunsfeld and Andrew Feustel, along with Michael Good and Michael Massimino worked on preparing for the next day's spacewalk, gathering tools and checking out the suits and equipment that would be used during the EVA.

The first EVA was performed by John Grunsfeld and Andrew Feustel on May 14, 2009 (7h 20m) to replace a wide field camera. They also swapped out three batteries and installed a mechanism for a spacecraft to capture for de-orbit at the end of the telescope's life.

The second EVA by Michael Massimino and Michael Good occured on May 15, 2009 (7h 56m) to change out three boxes, each containing two of the telescope's six gyroscopes, and three batteries.

The third EVA was again performed by John Grunsfeld and Andrew Feustel on May 16, 2009 (6h 36m) to install the Cosmic Origins Spectrograph, a device that will perform spectroscopy, the science of breaking up light into its individual components. The astronauts also conducted repairs to the Advanced Camera for Surveys, which has experienced power failures, causing two of the telescope's three observing channels to stop operating.

The fourth EVA by Michael Massimino and Michael Good was conducted on May 17, 2009 (8h 02m) to repair and upgrade the Space Telescope Imaging Spectrograph, which stopped working in 2004 due to power failures, and install a stainless steel blanket on Hubble's exterior. The blankets provide additional thermal protection for some equipment bays, replacing the existing multi-layer insulation that has degraded over time.

The fifth and final EVA by John Grunsfeld and Andrew Feustel was performed on May 18, 2009 (7h 02m) to replace a sensor needed for pointing the telescope and install another blanket on Hubble's exterior.

The landing was delayed two days due of bad weather in Florida.

STS-125 was the only planned shuttle mission after the Columbia accident to be launched into a low-inclination orbit that did not allow rendezvous with the International Space Station. Due to the inclination and other orbit parameters of Hubble, Atlantis would have been unable to use the International Space Station as a safe haven in the event of structural or mechanical failure. To preserve NASA's post Columbia requirement of having shuttle Launch On Need (LON) rescue capability, STS-400 was the flight designation given to the Contingency Shuttle Crew Support (CSCS) mission which would have been flown by Endeavour in the event Atlantis became disabled during STS-125. After Atlantis performed the late inspection and was cleared for re-entry, Endeavour was officially released from stand-by status on Thursday, May 21, 2009.