The transcription factor NF-κB is essential for survival of many cell types. However, cells can undergo apoptosis despite the concurrent NF-κB activation. It is unknown how the protection conveyed by NF-κB is overridden during apoptosis. We report here that IκB kinase (IKK) β was specifically proteolyzed by Caspase-3-related caspases at aspartic acid residues 78, 242, 373, and 546 during tumor necrosis factor (TNF)-α-induced apoptosis. Proteolysis of IKKβ eliminated its enzymatic activity, interfered with IKK activation, and promoted TNF-α killing. Point mutations that abrogate IKKβ proteolysis generated a caspase-resistant IKKβ mutant, which suppressed TNF-α-induced apoptosis. Thus, our study demonstrates that TNF-α-induced apoptosis requires caspase-mediated proteolysis of IKKβ.