The KRAS is an important and frequently mutated gene during colorectal carcinogenesis. The expression of miR-143 is often down-regulated and it might play an important role by targeting KRAS in colorectal cancer (CRC). The purpose of this study was to investigate the antitumor effects of miR-143 with an intermediate oncolytic adenovirus (Ad) in CRC. We constructed the recombinant virus Ad-ZD55-miR-143 and verified its expression by qPCR and western blot assays. Oncolytic potency of Ad-ZD55-miR-143 was determined by cytopathic effect assays using human SW480 CRC cells and L-02 normal liver cells. MTT and cell apoptosis assays were applied to explore the biological functions of Ad-ZD55-miR-143 within SW480 cells. Dual-luciferase reporter assays were performed to validate whether KRAS was regulated by miR-143. The expression level of KRAS was measured by qPCR and western blot assays. Results showed that infection of SW480 cells with Ad-ZD55-miR-143 induced high level expression of miR-143. Furthermore, Ad-ZD55-miR-143 significantly suppressed the viability of SW480 cells in a dose-dependent pattern, but did not influence L-02 cells. Ad-ZD55-miR-143 also inhibited cell growth and induced cell apoptosis in SW480 cells. Dual-luciferase assays indicated that KRAS was a direct target of miR-143, as subsequently demonstrated by qPCR and western blot analysis showing that infection of SW480 cells with Ad-ZD55-miR-143 resulted in the down-regulation of KRAS at both mRNA and protein levels. Taken together, the recombinant virus Ad-ZD55-miR-143 exhibited specific antitumor effects by targeting KRAS, and might be a promising agent for the treatment of CRC.