This study was undertaken to investigate the mechanism by which phenethyl isothiocyanate (PEITC), a natural compound from cruciferous vegetables, exhibits antitumor effect on prostate cancer cells. Cell proliferation, cell cycle, Western blot, gene transfer, and reporter assays were used to test the effects of PEITC on the growth and IL6/JAK/STAT3 pathway in prostate cancer. The result showed that PEITC significantly inhibited DU145 cell proliferation in a dose-dependent manner and induced the cell arrest at G2-M phase. PEITC inhibited both constitutive and IL-6-induced STAT3 activity in DU145 cells. IL-6-stimulated phosphorylation of JAK2, an STAT3 upstream kinase, was also attenuated by PEITC. Moreover, an antioxidant reagent, N-acetyl-L-cysteine (NAC) which suppresses reactive oxygen species (ROS) generation, reversed the early inhibitory effects of PEITC on cell proliferation, constitutive or IL-6-mediated JAK-STAT3 phosphorylation in PCa cells. Taken together, our data demonstrated that PEITC can inhibit the activation of the JAK-STAT3 signal-cascade in prostate cancer cells and the underlying mechanism may be partially involved with blocking cellular ROS production during the early stage of the signaling activation by IL-6.