Apoptin is a chicken anemia virus-derived, p53-independent, bcl-2-insensitive apoptotic protein with the ability to specifically induce apoptosis in various human tumor cells, but not in normal cells. To explore the use of apoptin in tumor gene therapy, we assessed a recombinant adenovirus expressing the apoptin protein (Ad-hTERTp-E1a-Apoptin) in order to determine its lethal and growth-inhibitory effects on PC-3 and RM-1 cells in vitro and its antitumor effect on solid tumors in vivo. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), acridine orange (AO)/ethidium bromide (EB), 4'-6-diamidino-2-phenylindole (DAPI), and Annexin V assays showed that Ad-hTERTp-E1a-Apoptin inhibited the proliferation of PC-3 and RM-1 cells in vitro by inducing apoptosis of prostate cancer cells, and that this inhibitory effect was dose and time-dependent. In the animal models, Ad-hTERTp-E1a-Apoptin significantly inhibited tumor growth and extended the lifespan of animals. Experimental results indicate that Ad-hTERTp-E1a-Apoptin has a potential application in tumor gene therapy.