Background & Aims: RNA interference (RNAi) is a powerful tool to silence gene expression. The adenoviral vector expressing small interfering RNA (siRNA) is highly effective in mammalian cells. However, its potential use as a therapeutic tool to target an oncogene specifically remains to be seen. We applied the adenovirus-delivered siRNA (AdSiRNA) to inhibit a hepatocellular carcinoma (HCC) oncogene, p28^GANK, in HCC cell lines and investigated its antitumor effects. Methods: The T7-RNA polymerase system was used to screen the specific target site. Double-strand oligonucleotide for transcription of short hairpin RNA was constructed into the adenoviral vector. Four HCC cell lines were infected with the RNAi-containing adenovirus. The RNAi effects on HCC were studied in cultured cells as well as in animal models. Results: p28 ^GANK expression was suppressed by up to 80% in HCC cells. Depletion of p28^GANK inhibited HCC cell growth and tumorigenesis, enhanced dephosphorylation of RB1, and decreased transcription activity of E2F-1 in HuH-7 cells. Furthermore, depletion of p28^GANK induced caspase-8- and caspase-9-mediated apoptosis of HCC cells. Finally, targeting p28^GANK by adenovirus injection inhibited the growth of established tumors in nude mice. Conclusions: This study shows that the T7-system screening-based AdSiRNA can be used successfully to silence an oncogene. We proved the therapeutic potential of AdSiRNA on the treatment of HCC by targeting p28^GANK. Our results indicate that p28^GANK may serve as a novel therapeutic target for treating HCC.