Stat3, a member of the signal transduction and activation of transcription (STAT) family, is a key signal transduction protein that mediates signaling by cytokines, peptide growth factors, and oncoproteins and is constitutively activated in numerous cancers including prostate. Previous studies demonstrated that constitutively activated Stat3 plays an important role in the development and progression of prostate cancer by promoting cell proliferation and protecting against apoptosis. The present study was designed to investigate the potential use of RNA interference to block Stat3 expression and activation and the effect on the growth of human prostate cancer cells. We identified a small interfering RNA (siRNA) specific for Stat3 and demonstrate that blockade of Stat3 activation by the Stat3 siRNA suppresses the growth of human prostate cancer cells and Stat3-mediated gene expression and induces apoptotic cell death. The Stat3 siRNA does not inhibit the proliferation nor induces apoptosis of Stat3-inactive human prostate cancer cells. In addition, the Stat3 siRNA inhibits the levels of androgen-regulated prostate specific antigen (PSA) expression in prostate cancer cells. These results demonstrate that targeting Stat3 signaling using siRNA technique may serve as a novel therapeutic strategy for treatment of prostate cancer expressing constitutively activated Stat3.