Infection of mammalian cells with viruses activates NF-κB to induce the expression of cytokines and chemokines and initiate an antiviral response. Here, we show that a vaccinia virus protein mimics the transactivation domain of the p65 subunit of NF-κB to inhibit selectively the expression of NF-κB-regulated genes. Using co-immunoprecipitation assays, we found that the vaccinia virus protein F14 associates with NF-κB co-activator CREB-binding protein (CBP) and disrupts the interaction between p65 and CBP. This abrogates CBP-mediated acetylation of p65, after which it reduces promoter recruitment of the transcriptional regulator BRD4 and diminishes stimulation of NF-κB-regulated genes CXCL10 and CCL2. Recruitment of BRD4 to the promoters of NFKBIA and CXCL8 remains unaffected by either F14 or JQ1 (a competitive inhibitor of BRD4 bromodomains), indicating that BRD4 recruitment is acetylation-independent. Unlike other viral proteins that are general antagonists of NF-κB, F14 is a selective inhibitor of NF-κB-dependent gene expression. An in vivo model of infection demonstrated that F14 promotes virulence. Molecular mimicry of NF-κB may be conserved because other orthopoxviruses, including variola, monkeypox and cowpox viruses, encode orthologues of F14.