In recent years, many have focused on the use of oncolytic virus capable of lysing cancer cells by a cancer-selective replication for a new treatment modality of cancer. In the present study, we used oncolytic adenovirus, AdCEAp/Rep, genetically modified to selectively replicate in CEA-expressing cancer cells, and investigated whether AdCEAp/Rep could induce selective cytotoxicity to CEA expressing cancer cells, and where the AdCEAp/Rep induced cytotoxic effect could be enhanced by 5 FU in using human gastric cancer cell lines, MKN45 (CEA-positive) and MKN74 (CEA-negative). The results showed that AdCEAp/Rep showed cytotoxicity against MKN45 in a dose-dependent manner, while it did not against MKN74. Furthermore, 5-FU remarkably enhanced AdCEAp/Rep-induced cytotoxicity against MKN45 by being added 3 days after adenovirus infection. These findings strongly suggest that AdCEAp/Rep may be applicable as a new therapeutic agent for clinical cancers expressing CEA in combination with chemotherapeutic agents such as 5-FU.