Purpose: Recent studies showed that oncolytic adenoviruses not only have capacity for destruction of tumors but also can be used as potential vectors to express therapeutic genes for therapy of cancer. However, better specificity and mode of transgene expression are required to improve the efficacy and safety if this vector is applied for clinical application. Experimental Design: In this study, we have created adenoviral replication-based transgene expression system by replacement of 6.7K/gp19K of E3 genes with EGFP and IL-24 genes so that expression of transgenes should be controlled by adenoviral E3 promoter. Transgene expression, viral replication capacity, and cytotoxicity have been studied in tumor and normal cells. Antitumor efficacy was evaluated in animal model with established tumor. Results: Our data showed that expression of IL-24 could be detected at 6 h and reached the maximal level at 48 h after infection in tumor cells. The expression level was 14 times higher than that induced by cytomegalovirus promoter. Low level of IL-24 could be detected in normal cells only until 72 h after infection. The substitution of 6.7K/gp19K of E3 genes with transgenes did not affect viral replication in tumor cells. Strong cytotoxicity was observed only in tumor cells after infection with AdCN205-IL-24. Treatment of the established tumors induced high level of local expression of IL-24 in tumor cells and resulted in tumor regression. Conclusions: Our data showed that AdCN205-IL-24 can provide potent and safe vector for the therapy of cancer. ©2008 American Association for Cancer Research.