The transcription factor nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) plays a critical role in host defense against viral infection by inducing the production of proinflammatory mediators and type I interferon. Consequently, viruses have evolved many mechanisms to block its activation. The poxvirus vaccinia virus (VACV) encodes numerous inhibitors of NF-κB activation that target multiple points in the signaling pathway. A derivative of VACV strain Copenhagen, called vv811, lacking 55 open reading frames in the left and right terminal regions of the genome was reported to still inhibit NF-κB activation downstream of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), suggesting the presence of one or more additional inhibitors. In this study, we constructed a recombinant vv811 lacking the recently described NF-κB inhibitor A49 (vv811ΔA49), yielding a virus that lacked all currently described inhibitors downstream of TNF-α and IL-1β. Unlike vv811, vv811ΔA49 no longer inhibited degradation of the phosphorylated inhibitor of κBα and p65 translocated into the nucleus. However, despite this translocation, vv811ΔA49 still inhibited TNF-α- and IL-1β-induced NF-κB-dependent reporter gene expression and the transcription and production of cytokines induced by these agonists. This inhibition did not require late viral gene expression. These findings indicate the presence of another inhibitor of NF-κB that is expressed early during infection and acts by a novel mechanism downstream of p65 translocation into the nucleus.