In addition to coordinating immune and inflammatory responses, NF-κB/Rel transcription factors control cell survival. The NF-κB antiapoptotic function is crucial to oncogenesis, cancer chemoresistance, and to antagonize tumor necrosis factor (TNF) receptor-induced killing. Recently, we have shown that the suppression of the c-Jun-N-terminal kinase (JNK) cascade is a pivotal protective mechanism by NF-κB, and that this suppression involves the upregulation of gadd45β/myd118. Induction of gadd45β by stress and cytokines requires NF-κB; however, the regulatory mechanisms underlying this induction are not known. Here, we report that, in HeLa cells, the NF-κB subunit RelA is sufficient to activate gadd45β expression, whereas Rel and p50 are not. Activation of gadd45β by RelA depends on three κB elements at positions -447/-438 (κB-1), -426/-417 (κB-2), and -377/-368 (κB-3) of the gadd45β promoter. Each of these sites binds to NF-κB complexes in vitro, and is required for optimal promoter transactivation. The data establish the direct participation of NF-κB in the regulation of Gadd45β, thereby providing important mechanistic insights into the control of apoptosis by the transcription factor.