The shuttle was due to have blasted off early on Wednesday, but hydrogen gas leaked from a vent line on its external fuel tank, officials said.
A previous leak postponed the shuttle's launch on Saturday.
The shuttle had been due to deliver part of a Japanese laboratory to the International Space Station (ISS).
"At 1.55am (05.55 GMT) launch managers called a scrub, cancelling today's planned launch of space shuttle Endeavour on its STS-127 mission," a Nasa statement said.
"Despite troubleshooting efforts, engineers were unable to achieve a decrease in the liquid hydrogenleak."
Spacewalks
The next ISS construction mission will not now take place until 11 July
Once it goes ahead, the astronauts will carry out five spacewalks to attach a permanent platform weighing 1.9 tonnes to the Japanese laboratory.
During the mission, the ISS will be a temporary home to 13 astronauts - the first time so many have stayed on the station at once.
Later on Wednesday a committee reviewing US plans for human space flight is holding a public hearing in Washington.
It is examining Nasa proposals to replace the shuttle fleet and the agency's plans for manned space flight.
President Obama has underlined the commitment of the US to return to the moon by the year 2020.
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