This study is to construct a lentiviral vector harbouring an RNA interference (RNAi) sequence that targets the gene encoding the human high-mobility group nucleosomal binding protein 1 (NSBP1); to study its role in inducing G(2)/M phase arrest and apoptosis in prostate cancer (PCa) DU145 cells; and to assess the effect of its knockdown on cell proliferation in vitro and in vivo. RNAi was applied to knock down NSBP1 expression in the PCa cell line DU145 by lentiviral plasmids producing an NSBP1 small hairpin RNA. After NSBP1 knockdown in DU145 cells, the growth rate of cells was analyzed by MTT, and G(2)/M cell cycle arrest and apoptosis were assessed using a FACScalibur flow cytometer. Tumour growth was assessed in nude mice. The mRNA and protein expression levels of NSBP1, cyclin B1 and Bcl-2 were analysed in vitro and in vivo by reverse-transcriptase polymerase chain reaction and Western blotting. Knockdown of NSBP1 resulted in a 22.6% decrease in the growth rate of cells compared with the PscNC lentivirus control cells at 96 h, decreased tumour growth in nude mice, and the induction of G2/M cell cycle arrest (8.78%) and apoptosis (2.19-fold). Consistent with the cell cycle arrest and apoptosis, the mRNA and protein expression levels of cyclin B1 and Bcl-2 were decreased. In conclusion, knockdown of NSBP1 causes a statistically significant inhibition of the in vitro and in vivo growth of the PCa cell line DU145. Growth suppression is at least partially due to NSBP1 knockdown-induced G2/M cell cycle arrest and apoptosis. The present data provide the evidence that the NSBP1 knockdown-induced G2/M phase arrest and apoptosis may result from negative regulation of cyclin B1 and Bcl-2 by NSBP1, with the resulting reduced expression of these proteins.