Forward Dragan Mrdja scored twice for the lively Serbs, by far the hungrier of the two South Africa-bound teams in a match for which neither coach had a full-strength side available.
"We did exactly what we knew we couldn't afford to by conceding an early goal," Japan captain Yuji Nakazawa told Reuters.
"We know we're going to face even more dangerous players at the World Cup in June. We just have to take this loss on the chin and move forwards."
Mrdja put Serbia ahead in the 15th minute with a clinical left-foot finish after bursting clear, 10 minutes after hitting the post from a similar position.
He smashed the ball home again from close range in the 23rd minute after Japan goalkeeper Seigo Narazaki had blocked his initial shot for his second goal in three internationals.
Midfielder Nemanja Tomic curled in a free kick on the hour mark, triggering jeers among the 46,000 crowd in Osaka which intensified as Japan trudged off after the final whistle.
ANTIC HEADACHE
"There was plenty of quality tonight for (Serbia coach Radomir) Antic to consider when he chooses his World Cup squad," Serbia assistant coach Radovan Curcic told reporters.
"The players showed they will soon be competing for places with the senior players."
The Japanese offered little to suggest they will achieve the semi-final target set by coach Takeshi Okada for the World Cup.
"I honestly thought we would be better than that," a glum Okada told reporters. "There wasn't the motivation there should be when you wear the Japan shirt. For that I apologise.
"We had 70 percent of the possession but did nothing with it. It has underlined how reliant we are on our top players."
Serbia were as solid in defence as they were menacing in attack, goalkeeper Zeljko Brkic making an astonishing fingertip save from Shinji Okazaki with the score at 1-0.
"We were poor. We had no rhythm," said Japan playmaker Shunsuke Nakamura. "We need to go back to basics."
Japan have been drawn alongside Netherlands, Denmark and Cameroon in Group E at the World Cup finals while Serbia face Germany, Australia and Ghana in Group D.
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