Launch from Cape Canaveral (KSC); landing at Cape Canaveral (KSC); ISS-MPLM Leonardo.

The primary payload was a Multi-Purpose Logistics Module loaded with supplies and equipment for the International Space Station. The mission also removed and replaced an ammonia tank assembly outside the station on the S1 truss. STS-131 furthermore carried several on-board payloads; this mission had the most payloads since STS-107.

Shortly after launch the shuttle Discovery's Ku-band antenna system, used as a radar dish during rendezvous operations and to transmit video and data to and from the ground through NASA communications satellites, suffered a malfunction of some sort. Loss of the antenna operations did not impact mission safety or success. Discovery made a safely rendezvous and dock with the station and successfully complete all of its planned mission objectives without use of the Ku-Band antenna, if needed.

Following a two day solo flight Discovery docked to the ISS on April 07, 2010. Joint operations with the ISS expedition 23 were performed.

On April 08, 2010 the Leonardo Multipurpose Logistics Module was relocated from Discovery's payload bay to a port on the Harmony node. The Italian built module's more than 17,000 pounds of cargo includes four experiment racks along with the final private crew quarters. This is the final roundtrip to the station for the 21-foot-long, 15-foot-diameter Leonardo. Once back on Earth, the module will be reconfigured with increased shielding on the outside for the STS-133 mission in September 2010 when it will be left on the station as a permanent module.

The first EVA by Richard Mastracchio and Clayton Anderson occured on April 09, 2010 (6h 27m). The crew inside used the station's robotic arm to remove a new ammonia tank from shuttle's payload bay and temporarily stow it on the station. Ammonia is used to move excess heat from inside the station to the radiators located outside. The spacewalkers then retrieved a seed experiment outside the Japanese laboratory. Next, the pair installed a grapple bar to the new ammonia tank on the station's truss. The pair also replaced a failed gyroscope that is part of the station's navigation system. They also accomplished several "get-ahead" tasks, removing 11 out of 12 p-clamps slated for a future spacewalk.

Meanwhile mission managers extended the flight by one day to enable a standard late inspection of the shuttle heat shield to occur while the shuttle is docked to the International Space Station.

The second EVA was performed by Richard Mastracchio and Clayton Anderson on April 11, 2010 (7h 26m). Using the station's arm, the astronauts removed the empty ammonia tank from the station's truss and temporarily stowed it on an equipment cart. The new tank then was installed and connected to the truss for use. The astronauts than ran into problems bolting the 1,700-pound structure down, however, put the astronauts well behind schedule and caused Mission Control to postpone hooking up the fluid lines to the new tank until a future EVA. Finally, after multiple attempts over an hour and a half, Clayton Anderson drove a recalcitrant bolt home to clear the problem. The station's arm then temporarily stowed the old tank on another part of the station's structure until the mission's third spacewalk.

The third and final EVA was conducted by Richard Mastracchio and Clayton Anderson on April 13, 2010 (6h 24m). First they finished the complicated change out of the large ammonia tank assembly. Using the station's arm, the crew moved the old tank into the shuttle's payload bay for return to Earth. The spacewalkers also removed the grapple bar from the old ammonia tank and stowed it on a spare parts platform. Finally Richard Mastracchio and Clayton Anderson retrieved debris shields from the Quest airlock to return to Earth. Due of the delays, the planned installing a plate and camera light on the Dextre robot, the removal of thermal covers on Dextre and replaceement of a burned out light on a truss camera were not carried out.

Meanwhile, engineers on the ground started to troubleshoot a stuck valve in a nitrogen tank assembly that's needed to pressurize a new ammonia tank installed on the space station by Discovery's astronauts. The ammonia tank was installed during three spacewalks by Mission Specialists Richard Mastracchio and Clayton Anderson.
Following some discussions the flight controllers have decided not to add a possible fourth spacewalk plus an additional one-day mission extension to the shuttle Discovery's mission to replace a nitrogen tank in the International Space Station's ammonia coolant system, concluding the system can safely operate for an extended period despite a stuck valve preventing normal pressurization.

On April 15, 2010 Stephanie Wilson and Naoko Yamazaki using the space station's robotic arm packed the Leonardo Multipurpose Logistics Module back into Discovery's cargo bay for return home.
The Leonardo cargo module was detached from the International Space Station late after a lengthy delay. It was parked overnight in a "low hover" position just above the shuttle's payload bay and the astronauts completed the installation next day morning.
The operation to move the Leonardo Multi-Purpose Logistics Module (MPLM) from the International Space Station back to space shuttle Discovery’s cargo bay for return to Earth has been delayed due to a problem with the mechanism that holds the MPLM in place.

Next Stephanie Wilson, Dorothy Metcalf-Lindenburger and Discovery Pilot James Dutton teamed up to begin the late inspection of the shuttle's thermal protection system. Working in shifts with some help from Commander Alan Poindexter and Naoko Yamazaki, they used Discovery's robotic arm and the orbiter boom sensor system to look at reinforced carbon-carbon of the wing leading edges and the nose cone, as well as and the heat-resistant tiles.
The inspection, scheduled for about seven hours, was finished almost three hours ahead of schedule. It was done while the shuttle was still docked so the images could be sent down by the station's high-data-rate system. Discovery's high-data-rate Ku band antenna is not working.

Due of bad weather in Florida the landing was delayed one day.