The IkappaB kinase (IKK) complex is a key regulator of signal transduction pathways leading to the induction of NF-kappaB-dependent gene expression and production of pro-inflammatory cytokines. It therefore represents a major target for the development of anti-inflammatory therapeutic drugs and may be targeted by pathogens seeking to diminish the host response to infection. Previously, the vaccinia virus (VACV) strain Western Reserve B14 protein was characterised as an intracellular virulence factor that alters the inflammatory response to infection by an unknown mechanism. Here we demonstrate that ectopic expression of B14 inhibited NF-kappaB activation in response to TNFalpha, IL-1beta, poly(I:C), and PMA. In cells infected with VACV lacking gene B14R (vDeltaB14) there was a higher level of phosphorylated IkappaBalpha but a similar level of IkappaBalpha compared to cells infected with control viruses expressing B14, suggesting B14 affects IKK activity. Direct evidence for this was obtained by showing that B14 co-purified and co-precipitated with the endogenous IKK complex from human and mouse cells and inhibited IKK complex enzymatic activity. Notably, the interaction between B14 and the IKK complex required IKKbeta but not IKKalpha, suggesting the interaction occurs via IKKbeta. B14 inhibited NF-kappaB activation induced by overexpression of IKKalpha, IKKbeta, and a constitutively active mutant of IKKalpha, S176/180E, but did not inhibit a comparable mutant of IKKbeta, S177/181E. This suggested that phosphorylation of these serine residues in the activation loop of IKKbeta is targeted by B14, and this was confirmed using Ab specific for phospho-IKKbeta.