Nin one binding protein (NOB1p), encoded by the NOB1 gene, is a crucial molecule in the maturation of the 20S proteasome and protein degradation. The present study evaluates whether NOB1 is an appropriate molecular target for cancer gene therapy. In two ovarian cancer cell lines, SKOV3 and HEY, NOB1 expression was knocked down by a lentiviral short hairpin RNA (shRNA) delivery system. The RNA interference (RNAi)-mediated the downregulation of NOB1 expression markedly reduced the proliferative and colony-formation ability of ovarian cancer cells. Additionally, NOB1 shRNA-expressing lentivirus-treated ovarian cancer cells tended to arrest in the G0/G1 phase. These results suggested that NOB1 may act as an oncogenic factor in ovarian cancer and could be a potential molecular target for ovarian cancer gene therapy.