Virotherapy with oncolytic viruses is a highly promising approach for cancer therapy. To improve further the therapeutic effect of oncolytic viruses, therapeutic genes have been incorporated into these types of vectors. In this study, we have inserted hTRAIL (approved gene symbol TNFSF10) into the ZD55 vector, which was based on deletion of the adenoviral E1B 55-kDa gene and could replicate in and lyse p53-deficient tumors. Our data shows that infection of colorectal carcinoma cells with ZD55-hTRAIL resulted in tumor cell death that was much greater than that induced by ZD55 vector or replication-defective adenovirus expressing hTRAIL. In contrast to these, ZD55-hTRAIL did not induce any cytopathic effect in normal cells. Treatment of established tumor with ZD55-hTRAIL resulted in dramatic inhibition of tumor growth in an animal model of colorectal carcinoma. However, when the established tumors were treated by coadministration of ZD55-hTRAIL and Ad-k5, we observed complete eradication of the established tumors in all animals treated with the combined therapy. This strong anti-tumor activity was due to the fact that two genes may act with compensative (or synergic) effect through different mechanisms to kill tumors. Therefore, targeting dual gene-virotherapy may be one of the best strategies for cancer therapy if two suitable genes are chosen.