Smac mimetics (SM) have been recently reported to kill cancer cells through the extrinsic apoptosis pathway mediated by autocrine tumor necrosis factor (TNF). SM also activates nuclear factor-kappaB (NF-kappaB). However, how SM induces NF-kappaB and the role of NF-kappaB in SM-induced cancer cell death has not been well elucidated. We found that effective blockage of NF-kappaB had no detectable effect on SM compound 3 (SMC3)-induced TNF secretion, suggesting that the induction of TNF by SMC3 is independent of NF-kappaB. Conversely, SMC3-induced NF-kappaB activation was found to be mediated by autocrine TNF because this effect of SMC3 was effectively inhibited when TNF was blocked with either a TNF neutralizing antibody or TNF small interfering RNA. In addition, although SMC3 dramatically reduced c-IAP1 level, it had marginal effect on c-IAP2 expression, TNF-induced RIP modification, NF-kappaB activation, and downstream antiapoptosis NF-kappaB target expression. Furthermore, blocking NF-kappaB by targeting IKKbeta or RelA substantially potentiated SMC3-induced cytotoxicity, suggesting that the NF-kappaB pathway inhibits SMC3-induced apoptosis in cancer cells. Our results show that through TNF autocrine, SM induces an IKKbeta-mediated NF-kappaB activation pathway that protects cancer cells against SM-induced apoptosis, and thus, NF-kappaB blockage could be an effective approach for improving the anticancer value of SM.