Nin one binding-1 protein (NOB1) is a kind of zinc protein involved in ribosome biogenesis and controlled proteolysis. To explore the function of NOB1 in human prostate malignancy, we analyzed the expression of NOB1 in prostate cancer and found that NOB1 was elevated in prostate cancer tissues compared to the adjacent normal tissues. Knockdown of NOB1 by lentivirus-shRNA inhibited the proliferation and colony-formation ability of PC-3 and DU145 prostate cancer cells. Cell cycle analysis showed that silencing of NOB1 caused G0/G1 phase arrest and a slight decrease in S phase (P < 0.05). Furthermore, knockdown of NOB1 significantly suppressed the mobility of PC-3 and DU145 prostate cancer cells (P < 0.05). Collectively, these findings suggested that NOB1 might be involved in tumorigenecity of prostate cancer, and could be a potential molecular target for prostate cancer gene therapy.