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IκB kinase (IKK) complex is a key regulator of NF-κB pathways. Signal-induced interaction of the IKKγ (NEMO) subunit with the C-terminal IKKγ/NEMO-binding domain (γBD) of IKKβ is an essential interaction for IKK regulation. Underlying regulatory mechanism(s) of this interaction are not known. Phosphorylation of γBD has been suggested to play a regulatory role for IKK activation. However, a kinase that phosphorylates γBD has not been identified. In this study, we used a C-terminal fragment of IKKβ as substrate and purified Polo-like kinase 1 (Plk1) from HeLa cell extracts by standard chromatography as a γBD kinase. Plk1 phosphorylates serines 733, 740, and 750 in the γBD of IKKβ in vitro. Phosphorylating γBD with Plk1 decreased its affinity for IKKγ in pulldown assay. We generated phosphoantibodies against serine 740 and showed that γBD is phosphorylated in vivo. Expressing a constitutively active Plk1 in mammalian cells reduced tumor necrosis factor (TNF)-induced IKK activation, resulting in decreased phosphorylation of endogenous IκBα and reduced NF-κB activation. To activate endogenous Plk1, cells were treated with nocodazole, which reduced TNF-induced IKK activation, and increased the phosphorylation of γBD. Knocking down Plk1 in mammalian cells restored TNF-induced IKK activation in nocodazole-treated cells. Activation of Plk1 inhibited TNF-induced expression of cyclin D1. In cells in which Plk1 was knocked down, TNFα increased expression of cyclin D1 and the proportion of cells in the S phase of the cell cycle. Taken together, this study shows that phosphorylation regulates the interaction of γBD of IKKβ with IKKγ and therefore plays a critical role for IKK activation. Moreover, we identify Plk1 as a γBD kinase, which negatively regulates TNF-induced IKK activation and cyclin D1 expression, thereby affecting cell cycle regulation. Untimely activation of cyclin D1 by TNFα can provide a potential mechanism for an involvement of TNFα in inflammation-induced cancer.