The CD95 (Apo1/Fas)/CD95 ligand system plays pivotal roles in various aspects of immune regulation and function by triggering apoptosis. Besides the apoptosis signaling pathway, CD95 ligation also induces the activation of NF-kappaB. Previous studies suggest that IkappaB kinase (IKK) may be a key player in cell survival by mediating NF-kappaB activation. However, the roles of IKK in CD95 ligation-mediated apoptosis and NF-kappaB activation are still not clear. In this report, we show that expression of the caspase-resistant uncleavable IKKbeta (UCIKKbeta) mutant suppressed CD95 ligation-mediated cell death in HeLa cells. Furthermore, CD95 ligation induced much more cell death in IKKbeta-/- murine embryonic fibroblasts (MEFs) than in wild type MEFs, despite that IKK was only marginally activated upon CD95 ligation. Pretreatment of HeLa cells with a specific IKK inhibitor NEMO-binding domain (NBD) peptide blocked CD95 ligation-induced NF-kappaB transcriptional activity. And UCIKKbeta enhanced the basal NF-kappaB activity, and consequently led to higher NF-kappaB activity upon CD95 ligation in HeLa cells. Therefore, IKK antagonizes CD95 ligation-mediated apoptosis by regulating NF-kappaB activity.