Interferon gamma (IFNgamma) is regarded as a potent antitumor agent, but therapy with IFNgamma is hampered by its short half-life and significant side effects. We developed a replication defective adenovirus carrying the human IFNgamma gene and evaluated the effects of adenovirus-mediated IFNgamma (Ad-IFNgamma) gene transfer on human prostate cancer cell lines in vitro and on xenografts in vivo. Our results showed infection of prostate cancer cells with Ad-IFNgamma led to production of an active cytokine and resulted in an antiproliferative effect on the prostate cancer cells. Intratumoral injection of Ad-IFNgamma significantly inhibited the growth of DU-145 cell xenografts in vivo, while no significant toxicity effect was observed. RT-PCR analysis indicated transgene expression mainly enriched in tumors in vivo, and slightly distributed in livers. These findings suggest adenovirus-mediated IFNgamma gene transfer is a promising approach in the treatment of advanced prostate cancer.