The present study reports a novel response to sulforaphane, a highly promising anticancer constituent of several edible cruciferous vegetables, in PC-3 and LNCaP human prostate cancer cells involving induction of autophagy. Exposure of PC-3 and LNCaP cells to sulforaphane resulted in several specific features characteristic of autophagy, including appearance of membranous vacuoles in the cytoplasm as revealed by transmission electron microscopy and formation of acidic vesicular organelles as revealed by fluorescence microscopy following staining with the lysosomotropic agent acridine orange. The sulforaphane-induced autophagy was associated with up-regulation, processing, and recruitment to autophagosomes of microtubule-associated protein 1 light chain 3 (LC3), which is a mammalian homologue of the yeast autophagy regulating protein Apg8/Aut7p. Treatment of cells with a specific inhibitor of autophagy (3-methyladenine) attenuated localization of LC3 to autophagosomes but exacerbated cytosolic release of cytochrome c as well as apoptotic cell death as revealed by analysis of subdiploid fraction and cytoplasmic histone-associated DNA fragmentation. In conclusion, the present study indicates that induction of autophagy represents a defense mechanism against sulforaphane-induced apoptosis in human prostate cancer cells. To the best of our knowledge, the present study is the first published report to convincingly document induction of autophagy by an isothiocyanate class of dietary chemopreventive agent.