Osteopontin (OPN) has been recognized as a significant cytokine in the processes of tumorigenicity, tumor progression and metastasis in many types of human cancer. However, the functions of OPN in prostate cancer remain poorly understood. To investigate the function of OPN in human prostate cancer, the growth of prostate cancer PC-3 cells was examined following OPN down-regulation by RNA interference (RNAi). PC-3 cells were transfected by two constructs containing short interfering RNAs designed to cleave two different regions of OPN mRNA. The expression of OPN in the transfected cells was markedly inhibited by RNAi at the mRNA and protein levels. Cell growth was retarded and S-phase arrest and apoptosis were observed in the transfected cells. The number and size of the colonies of the transfected cells in soft agarose were markedly decreased, as compared with those of the control cells. From these results, we conclude that the selective down-regulation of OPN expression by RNAi may lead to S-phase arrest, apoptosis and a decline in the malignant phenotype in PC-3 cells, suggesting that OPN plays a significant role in the growth of prostate cancer and may be a potential therapeutic target.