Purpose: Recent studies have indicated that short hairpin RNA (shRNA) driven by RNA polymerase (Pol) II promoters can be transcribed into precursor mRNAs together with transgenes. It remains unclear, however, whether coexpression of shRNA and transgene from a single promoter is feasible for cancer therapy. Experimental Design: In this study, we generated novel adenoviral vectors that permitted coexpression of shRNA against cyclooxygenase-2 (COX-2) and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) therapeutic gene from a cytomegalovirus promoter to evaluate whether silencing of COX-2 could increase the sensitivity of hepatocellular carcinoma to TRAIL. Results: Our data showed that adenovirus vector Ad-TM, in which the shRNA was inserted into the 3′ untranslated region of the TRAIL gene, not only significantly suppressed COX-2 expression, but also expressed a high level of TRAIL. Moreover, infection with Ad-TM resulted in significant cytotoxicity in hepatocellular carcinoma cell lines. In contrast, it had no effect on normal liver cell line. Impressively, treatment of the established hepatocellular carcinoma tumors with Ad-TM resulted in complete tumor regression. This potent antitumor activity induced by Ad-TM was due to strong inhibition of COX-2 and high expression of TRAIL. Furthermore, using the shRNA and transgene coexpression adenovirus system, we showed that silencing of COX-2 increased the sensitivity of hepatocellular carcinoma to TRAIL through inhibition of Bcl-2 and Bcl-w. Conclusion: This study indicated that adenovirus carrying shRNA and transgene expressed from a single promoter represented a potent approach for cancer therapy. ©2010 AACR.