Diallyl disulfide (DADS) induced apoptosis through the caspase-3 dependent pathway in leukemia cells was earlier reported from this laboratory. In this study, we investigated the involvement of Ca2+ in DADS-induced apoptotic cell death of HCT-15, human colon cancer cell line. DADS induced the elevation of cytosolic Ca2+ by biphasic pattern; rapid Ca2+ peak at 3 min and following slow and sustained elevation till 3 h after the addition of DADS. Production of H2O2 was also observed with its peak value at 4 h. Apoptotic pathways including the sequence of caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation by DADS were completely blocked by various inhibitors such as specific caspase-3 inhibitor, free radical scavenger, and intracellular Ca2+ chelator. N-acetylcystein and catalase treatment prevented the accumulation of H2O2 and later caspase-3 dependent apoptotic pathway. However, these radical scavengers did not block the elevation of intracellular Ca2+. Treatment of cells with 1,2-bis(2-aminophenoxyethane)-N,N,N-tetraacetic acid tetralkis -acetoxymethylester (BAPTA-AM), cellular Ca2+ chelator, resulted in a complete blockage of the caspase-3 dependent apoptotic pathway of HCT-15 cells. It abolished the elevation of intracellular Ca2+, and furthermore, completely inhibited the production of H2O2. These results indicate that cytosolic Ca2+ elevation is an earlier signaling event in apoptosis of HCT-15 cells. Collectively, our data demonstrate that DADS can induce apoptosis in HCT-15 cells through the sequential mechanism of Ca2+ homeostasis disruption, accumulation of H2O2, and resulting caspase-3 activation.