Tumor necrosis factor-α (TNF-α) is a cytokine that has an important role in immunity and inflammation by inducing cellular responses such as apoptosis. The transcription factor nuclear factor-κB (NF-κB) can paradoxically suppress and promote apoptosis in response to TNF-α. In this study, we found that p53 upregulated modulator of apoptosis (PUMA), a p53 downstream target and a BH3-only Bcl-2 family member, is directly regulated by NF-κB in response to TNF-α. TNF-α treatment led to increases in PUMA mRNA and protein levels in human colon cancer cells. The induction of PUMA was p53 independent, and mediated by the p65 component of NF-κB through a κB site in the PUMA promoter. The apoptotic effect of PUMA induction by TNF-α was unmasked by depleting the antiapoptotic protein Bcl-X L. In mice, PUMA was also induced by TNF-α in an NF-κB-dependent manner. TNF-α-induced apoptosis in a variety of tissues and cell types, including small intestinal epithelial cells, hepatocytes, and thymocytes, was markedly reduced in PUMA-deficient mice. Collectively, these results demonstrated that PUMA is a direct target of NF-κB and mediates TNF-α-induced apoptosis in vitro and in vivo.