The transcription factor NF-?B is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of I?Ba. Here we describe an inhibitor of NF-?B from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-?B activation by molecular mimicry and contains a motif conserved in I?Ba which, in I?Ba, is phosphorylated by IKKß causing ubiquitination and degradation. Like I?Ba, A49 binds the E3 ligase ß-TrCP, thereby preventing ubiquitination and degradation of I?Ba. Consequently, A49 stabilised phosphorylated I?Ba (p-I?Ba) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the I?Ba-like motif of A49 abolished ß-TrCP binding, stabilisation of p-I?Ba and inhibition of NF-?B activation. Remarkably, despite encoding nine other inhibitors of NF-?B, a VACV lacking A49 showed reduced virulence in vivo.