Benzyl isothiocyanates (BITC), a member of the isothiocyanate (ITC) family, inhibits cell growth and induces apoptosis in many types of human cancer cell lines. The present study investigated mechanisms underlying BITC-induced apoptosis in A375.S2 human melanoma cancer cells. To observe cell morphological changes and viability, flow cytometric assays, cell counting, and a contrast-phase microscopic examination were carried out in A375.S2 cells after BITC treatment. Cell cycle distribution and apoptosis were assessed with the analysis of cell cycle by flow cytometric assays, DAPI staining, propidium iodide (PI), and annexin V staining. Apoptosis-associated factors such as reactive oxygen species (ROS) formation, loss of mitochondrial membrane potential (ΔΨ(m)), intracellular Ca(2+) release, and caspase-3 activity were evaluated by flow cytometric assays. Abundance of cell cycle and apoptosis associated proteins was determined by Western blotting. AIF and Endo G expression was examined by confocal laser microscope. Results indicated that (1) BITC significantly reduced cell number and induced cell morphological changes in a dose-dependent manner in A375.S2 cells; (2) BITC induced arrest in cell cycle progression at G(2)/M phase through cyclin A, CDK1, CDC25C/Wee1-mediated pathways; (3) BITC induced apoptosis and increased sub-G(1) population; and (4) BITC promoted the production of ROS and Ca(2+) and loss of ΔΨ(m) and caspase-3 activity. Furthermore, BITC induced the down-regulation of Bcl-2 expression and induced up-regulation of Bax in A375.S2 cells. Moreover, BITC-induced cell death was decreased after pretreatment with N-acetyl-l-cysteine (NAC, a ROS scavenger) in A375.S2 cells. In conclusion, the results showed that BITC promoted the induction of G(2)/M phase arrest and apoptosis in A375.S2 human melanoma cells through ER stress- and mitochondria-dependent and death receptor-mediated multiple signaling pathways. These data suggest that BITC has potential as an agent for the treatment of melanoma.