The classical nuclear factor kappa B (NF-κB) signaling pathway is an important regulator of inflammation and innate immunity that is activated by a wide variety of stimuli, including virus infection, tumor necrosis factor alpha (TNF-α), and interleukin 1β (IL-1β). Poxviruses, including vaccinia virus (VV) and ectromelia virus, encode multiple proteins that function in immune evasion. Recently, a growing number of genes encoded by poxviruses have been shown to target and disrupt the NF-κB signaling pathway. To determine if additional gene products that interfere with NF-κB signaling existed, we used a vaccinia virus deletion mutant, VV811, which is missing 55 open reading frames lacking all known inhibitors of TNF-α-induced NF-κB activation. Immunofluorescence analysis of HeLa cells treated with TNF-α and IL-1β revealed that NF-κB translocation to the nucleus was inhibited in VV811-infected cells. This was further confirmed through Western blotting of cytoplasmic and nuclear extracts for NF-κB. Additionally, VV811 infection inhibited TNF-α-induced IκBα degradation. In contrast to vaccinia virus strain Copenhagen (VVCop)-infected cells, VV811 infection resulted in the dramatic accumulation of phosphorylated IκBα. Correspondingly, coimmunoprecipitation assays demonstrated that the NF-κB-inhibitory IκBα-p65-p50 complex was intact in VV811-infected cells. Significantly, cells treated with 1-β-d-arabinofuranosylcytosine, an inhibitor of poxvirus late gene expression, demonstrated that an additional vaccinia virus late gene was involved in the stabilization of IκBα. Overall, this work indicates that unidentified inhibitors of NF-κB exist in vaccinia virus. The complex inhibition of NF-κB by vaccinia virus illustrates the importance of NF-κB activation in the antiviral response.