Ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis, was found to increase in cancer cells, especially prostate cancers. Some chemotherapeutic agents aimed to decrease ODC expression showed inhibitory effects on cancer cells. In this study, we examined the effect of adenoviral-transduced antisense ODC on prostate cancer cells. An adenovirus carrying antisense ODC (rAd-ODC/Ex3as) was infected to prostate cancer cells PC-3 and LNCap. Expression of ODC and concentration of polyamines in cells were determined by Western blotting and HPLC. MTT (3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay was used to analyze the effect on cell growth. Cell cycle was evaluated by FCM and cellular invasion by Matrigel invasion assay. A nude mouse xenograft model was used to examine tumorigenicity. Expression of ODC in PC-3 and LNCap cells were reduced to 45 and 59%, and three polyamines were also decreased by the rAd-ODC/Ex3as treatment. Consequently, cell growth was substantially inhibited and cell cycle arrested at G1 phase. Matrigel invasion assay showed relatively low invasion. Marked suppression of tumor formation was observed in the xenograft model. This study suggests that rAd-ODC/Ex3as has the antitumor effect on the human prostate cancer cells.