Nuclear factor (NF)-kB transcription factors are the master regulators of a variety of cellular processes, particularly immune and inflammatory responses. Activation of the IkB kinase β (IKKβ) is a central event in the nuclear factor-kB mediated canonical pro-inflammatory pathway, which has been indicated by numerous studies to be regulated by oligomerization-mediated trans auto-phosphorylation of the IKKβ kinase. The activation of the IKKβ kinase entails phosphorylation on two key serine residues S177 and S181, which are present on the IKKβ activation segment. This confers IKKβ kinase activity to phosphorylate downstream substrates such as the IkB proteins. In addition, its activation requires the interaction with NF-kB essential modifier (NEMO). Interestingly, some viruses encode proteins that inhibit NF-kB mediated anti-viral immune response by targeting the IKKβ kinase. B14 is a vaccinia viral protein that inhibits IKKβ activation through direct interaction. Using GST pull-down assay, we have mapped the binding interface on both the B14 and IKKβ proteins. B14 binds to both the kinase domain (KD) and the scaffold and dimerization domain (SDD) of IKKβ. Molecular docking modeling studies have identified key interface residues in both IKKβ and B14, which were further confirmed by mutational studies. This interaction causes a steric hindrance that blocks the direct contact between the kinase domains of IKKβ in the IKK complex, which contains both IKKβ and NEMO. During trans auto-phosphorylation of the IKKβ kinase, the activation segment of a kinase domain needs to interact with the active site of another neighbor kinase domain for trans phosphorylation and activation. As such, the B14-binding hinders IKKβ trans auto-phosphorylation and activation, which eventually inhibits NF-kB signaling.