Benzyl isothiocyanate (BITC) in cruciferous plants, which are part of the human diet, has been shown to induce apoptosis in various types of cancer. In this study, we show that BITC effectively suppresses the growth of cultured human prostate cancer cells (CRW-22Rv1 and PC3) by causing mitochondrial membrane potential loss, caspase 3/7 activation and DNA fragmentation. Furthermore, BITC induces ROS generation in these cells. The induction of apoptosis by BITC was significantly attenuated in the presence of N-acetylcysteine (NAC) and catalase (CAT), well-studied ROS scavengers. The induction of autophagy in BITC-treated cells were also diminished by the application of NAC or CAT. In addition, BITC-induced apoptosis and autophagy were both enhanced by the pretreatment of catalase inhibitor, 3-Amino-1,2,4-triazole (3-AT). Pretreatment with specific inhibitors of autophagy (3-methyladenine or bafilomycin A1) or apoptosis (Z-VAD-FMK) reduced BITC-induced autophagy and apoptosis, respectively, but did not abolish BITC-induced ROS generation. In conclusion, the present study provides evidences that BITC caused prostate cancer cell death was dependent on the ROS status, and clarified the mechanism underlying BITC-induced cell death, which involves the induction of ROS production, autophagy and apoptosis, and the relationship between these three important processes.