The massacre was "an atrocity committed by Japanese militarism during the war of aggression in China," and is backed up by "ironclad evidence," spokesman Kong Quan said on the ministry's Internet site.
"The international community has already made its final decision about this matter. Any attempts to distort or deny history would be futile," he said.
He made the remark in response to comments by Takami Eto, leader of the third-largest faction in Prime Minister Koizumi's Liberal Democratic Party.
Estimates by historians that Japanese troops killed 300,000 people in the so-called Rape of Nanking were "a big lie", Eto said on Saturday.
Eto, a 78-year-old three-time cabinet minister, has talked himself into trouble in the past when commenting on Japan's history.
He angered Koreans in 1997 by comparing Japan's annexation of Korea in 1910 to a "merger between two towns".
He reportedly touched on that theme during his speech on Saturday in the city of Fukui, saying that Japan's occupation of Korea between 1910 and 1945 should not be considered colonialism because both sides signed a treaty formalizing annexation.
A South Korean Foreign Ministry official said yesterday he was disappointed. "Our government want to make it clear that such remarks will not help South Korea-Japan relations," the official said.
"We once again emphasize that without Japan's correct understanding of history, it is difficult to sincerely develop relations between the two countries."