In the present study we assessed the contribution of acidic sphingomyelinase (ASMase), a ceramide generating enzyme, in tumor necrosis factor (TNF)-mediated apoptosis in human colon HT-29 cells. TNF induced apoptosis in HT-29 cells in a time- and dose-dependent fashion. Downregulation of the active endogenous ASMase form prevented TNF-stimulated ASMase activity and apoptosis. Furthermore, inhibition of glucosylceramide synthase, which blunted TNF-stimulated GD3 levels, abolished TNF-mediated cell death. Immunocytochemical staining revealed the co-localization of GD3 with mitochondria induced by TNF. The knockdown of targeted GD3 synthase by antisense expression vector protected HT-29 cells against TNF-induced cell death. Thus, ASMase plays a key role in TNF-induced cell death in human colon epithelial cells possibly through GD3 generation.