The fully loaded vessel -- carrying more than 1,580 kilograms of science samples -- disengaged from the space station's robotic arms as planned at 1326 GMT above the ocean near southern Australia, according to video streamed live on NASA television.
The cargo ship had spent 28 days at the orbiting outpost, after arriving on April 20.
It is expected to land in the Pacific Ocean six hours after undocking, at 1902 GMT, some 480 kilometres from the Mexican coast.
NASA is relying on SpaceX and other private firms to replace the Space Shuttles, which were finally retired in July 2011, and had brought supplies and astronauts to and from the space station.
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SpaceX has a USD 1.6 billion contract with NASA for a total of 12 of supply missions.
The US space agency has a second USD 1.9 billion contract to supply the space station with Orbital Sciences Corporation, whose Cygnus capsule completed its first delivery in January 2014.
That would end the US dependence on Russia's Soyuz capsules to transport US astronauts to and from the space station, at a cost of USD 70 million a seat.