High mobility group box protein 1 (HMGB1), transcriptional activity regulatory protein is associated with most cancers including prostate cancer. To investigate the effects of down-regulation of HMGB1 expression, we have transfected LNCaP cells with four short hairpin RNA (shRNA) targeting HMGB1 plasmid vectors. Transfection with the four shRNAs efficiently and specifically reduced the HMGB1 expression in LNCaP cells. The gene silencing effects on HMGB1 expression were subsequently confirmed by RT-PCR and immunoblotting analyses. Down-regulation of HMGB1 expression resulted in the inhibition of cell growth in LNCaP prostate cancer cells and the decreased cell number was due to transfected cells undergoing apoptosis via caspase-3-dependent pathways. These findings suggest that HMGB1 is critical for the survival of prostate cancer cells and targeted knockdown of HMGB1 mRNA can be used as a strategy to kill prostate cancer cells. Our findings may have some potential therapeutic relevance for treating prostate cancer.