The purpose of this study is to evaluate possible antitumor activity of a dual-regulated oncolytic adenovirus carrying the TAp63 gene on colorectal cancer. The recombinant virus Ad-survivin-ZD55-TAp63 was constructed by inserting the TAp63 gene into the dual-regulated pshuttle-survivin-ZD55 vector. RT-PCR and western blot assays were used to verify the recombinant virus Ad-survivin-ZD55-TAp63. Crystal violet staining was carried out to detect the cytopathic effect of Ad-survivin-ZD55-TAp63 in human colorectal cancer cell line HCT-116 and normal liver cell line L-O2. MTT and cell apoptosis assays were applied to explore the biological functions of Ad-survivin-ZD55-TAp63 within HCT116 cells. To further identify the antitumor effects of Ad-survivin-ZD55-TAp63 on HCT116 xenograft in BALB/C nude mice, tumor volumes were calculated and tumor tissues from the xenograft models were examined by TUNEL assays. The results showed that Ad-survivin-ZD55-TAp63 was successfully constructed, and could selectively replicate in HCT116 cells without significant toxicity to L-02 cells. Furthermore, Ad-survivin-ZD55-TAp63 dose- and time-dependently inhibited cell proliferation and induced cell apoptosis in vitro. In HCT116 xenograft models, intratumoral injection of Ad-survivin-ZD55-TAp63 significantly suppressed tumor growth and caused tumor cell apoptosis. Therefore, these results suggest that the recombinant virus Ad-survivin-ZD55-TAp63 exhibits specific antitumor effects, and may be used in the future for the treatment of colorectal cancer.