Curcumin exhibits anti-cancer properties manifested by activation of pro-apoptotic signaling. We have demonstrated earlier that apoptosis of HL-60 human leukemia cells induced by curcumin is controlled by ceramide generated by neutral sphingomyelinase (nSMase) which contributes to sphingomyelin synthase (SMS) inhibition favoring accumulation of ceramide in cells. Here we report that the activity of nSMase, ceramide accumulation and death of HL-60 cells are inhibited by overexpression of Bcl-xL or Bcl-2 proteins, while down-regulation of nSMase interferes with degradation of Bcl-2 but not Bcl-xL. Activation of nSMase in curcumin-treated cells requires the activity of apoptosis initiator caspase-8 and executioner caspase-3, whereas nSMase depletion prevents activation of caspase-3, but not caspase-8. These data place nSMase activation downstream of caspase-8 and Bcl-xL and indicate a mutual regulation between nSMase and caspase-3 activity on one hand, and Bcl-2 level on the other hand in curcumin-treated cells. The activation of nSMase and ceramide accumulation also depended on the depletion of glutathione. The depletion of glutathione required the activity of caspase-8 and caspase-3 as well as the down-regulation of Bcl-2 and Bcl-xL. Together, the data indicate a crosstalk among Bcl-2, Bc-xL, caspases and glutathione during curcumin-induced apoptosis and point to the superior role of caspase-8 activity, Bcl-xL down-regulation and glutathione depletion in the pro-apoptotic cascade leading to nSMase activation and generation of ceramide.