The transcription factor NF-kB plays important roles in inflammation and cell survival. NF-kB is composed of homodimeric and heterodimeric complexes of Rel/NF-kB family members, including p65 (RelA), c-Rel (Rel), RelB, NF-kB1/p50 and NF-kB2/p52. Here we report the identification and characterization of a novel ZU5 and death domain containing protein designated as ZUD. In reporter gene assays, overexpression of ZUD inhibited NF-kB-dependent transcription induced by TNF, IL1 and their downstream signaling proteins. Gel shift assays indicated that overexpression of ZUD inhibited binding of NF-kB to its target sequence. ZUD is a cytoplasmic protein and coimmunoprecipitation assays indicated that ZUD interacted with the NF-kB subunit p105 and transactivator p65. Consistent with its role in inhibition of NF-kB-dependent transcription, ZUD sensitized cells to apoptosis induced by TNF and TRAIL. Our findings suggest that ZUD is an inhibitor of NF-kB activation and this protein may provide an alternative regulatory mechanism of NF-kB-mediated transcription.