Poxviruses have evolved various strategies to inhibit cytoplasmic events leading to activation of the Nuclear Factor-κB (NF-κB) signaling pathway, with individual viruses often encoding for multiple NF-κB inhibitors. Here, the novel orf virus (ORFV)-encoded protein ORFV002 was shown to inhibit nuclear events regulating NF-κB transcriptional activity. ORFV002 expression in cell cultures significantly decreased wild-type virus-, tumor necrosis factor alpha (TNF-α)-, and lipopolysaccharide (LPS)-induced NF-κB-mediated gene expression. Expression of ORFV002 in cells, while not affecting phosphorylation or nuclear translocation of NF-κB-p65, markedly decreased TNF-α- and wild-type virus-induced acetylation of NF-κB-p65, a p300-mediated nuclear modification of NF-κB-p65 that regulates its transactivating activity. ORFV002 was shown to colocalize and interact with NF-κB-p65, and its expression in cell cultures resulted in a reduced interaction of NF-κB-p65 with p300, suggesting that ORFV002 functions interfering with NF-κB-p65/p300 association. Deletion of ORFV002 from the OV-IA82 genome had no significant effect on ORFV pathogenesis in sheep, indicating that ORFV002 is non essential for virus virulence in the natural host. This represents the first description of a nuclear inhibitor of NF-κB encoded by a poxvirus.