Beijing has called on Japanese authorities to avoid creating "artificial" incidents around the group of disputed, uninhabited islands in the East China Sea, known as the Senkaku islands in Japan and the Diaoyu islands in China.
The officials say the submarine again entered Japan's contiguous zone on Thursday morning off Taisho Island of the Senkaku Islands.
"This behavior unilaterally raises tensions, and we are seriously concerned about it", Defense Minister Itsunori Onodera told the press.
The government of China should refrain from military intrigues and the expression of emboldened statements about territorial waters that belong to Japan.
The Maritime Self-Defense Force spotted the submarine traveling in the contiguous zone east-northeast of Miyakojima, an island in Okinawa, on Wednesday afternoon.
Chinese officials defended the move Thursday, saying that the Senkakus are China's territory and that the incident was instigated by two Japanese navy vessels.
Global law requires submarines to surface and hoist the national flag when navigating through territorial waters of other nations, but not in contiguous zones.
Sugiyama "issued a protest by expressing serious concerns and strongly requested China not to interfere with the flow of improving Japan-China relations", the foreign ministry in Tokyo said in a statement. The sub exited the contiguous zone and headed toward the Senkakus on Thursday morning.
A Chinese frigate was also observed Thursday entering the zone, prompting Japan to protest that action. The Chinese ship did not enter Japanese territorial waters.
The warship and the submarine had left the waters by the afternoon, the defence ministry said in a statement.
Following the latest incident, Vice Foreign Minister Shinsuke Sugiyama summoned Chinese Ambassador to Japan Cheng Yonghua to the ministry and lodged a protest with him.
China's timing is equally unusual given the fact that Japan is hoping to improve relations between both nations.
Japan and China have always been at loggerheads over the tiny, uninhabited islands in the East China Sea.
China's defense ministry said that a Chinese missile frigate had responded to two Japanese ships entering its claimed waters but that the ships had now left.