We previously constructed OBP-301 (Telomelysin, a telomerase-specific replication-competent adenovirus with human telomerase reverse transcriptase (hTERT) promoter), which showed a strong anticancer effect by inducing cell lysis of human non-small cell lung cancer and colorectal cancer cells. To investigate the utility of OBP-301 for prostate cancer treatment, we herein evaluate the cell killing and antitumor effects. First, in vitro hTERT-specific adenovirus transduction in human prostate cancer cells (LNCaP, PC3, DU145) was confirmed using OBP-401 (Telomelysin-green fluorescent protein (GFP)). There was no detectable GFP transduction in the human prostate normal cells (PrEC, PrSC). Consistently, the cell-killing effect of OBP-301 was observed only in the cancer cells. Second, using an in vivo subcutaneous LNCaP tumor model in nude mice, we demonstrated that three intratumoral OBP-301 injections (10(7) PFU per tumor x 3 days) were sufficient to eradicate the detectable LNCaP prostate tumor. We also demonstrated that the ispilateral treatment with OBP-301 significantly suppressed contralateral LNCaP tumor growth in both sides of the tumor model. Histological and immunohistochemical analyses revealed diffuse oncolytic degeneration and adenoviral E1A protein expression in both sides of the tumors. Therefore, in situ OBP-301 administration could be a promising therapeutic strategy against prostate cancer and its metastatic lesions.