Prostate-specific membrane antigen is a type II membrane protein with folate hydrolase activity produced by prostatic epithelium. It has been demonstrated that prostate-specific membrane antigen over-expression may be correlated with prostate cancer, particularly in advanced cancer. The aim of the current study was to explore the possibility of prostate-specific membrane antigen as a therapeutic target for the treatment of prostate cancer. To address this problem, lentivirus-mediated small interfering RNA was employed to reduce endogenous prostate-specific membrane antigen expression in prostate cancer cell lines—LNCaP and DU-145. Then, the tumorigenesis, migration ability and invasiveness of prostate-specific membrane antigen-reduced prostate cancer cell lines were also examined. The prostate-specific membrane antigen expression in LNCaP and DU-145 cells was persistently and markedly reduced by lentivirus-mediated RNA interference. Down-regulation of prostate-specific membrane antigen expression significantly suppressed the growth rates of LNCaP and DU-145 cells. Moreover, the specific down-regulation arrested cells in G0/G1 phase of cell cycle. Furthermore, we also observed that the silence of prostate-specific membrane antigen could decrease the migration ability and the invasiveness of LNCaP and DU-145 cells. Our investigation demonstrated that lentivirus-mediated RNA interference silencing targeting prostate-specific membrane antigen might reduce the proliferation, and induce potent antitumor activity of LNCaP and DU-145 cells. Prostate-specific membrane antigen has considerable potential as a new therapeutic target for the treatment of prostate cancer.