Transcription factor NF-κB governs the expression of multiple genes involved in cell growth, immunity, and inflammation. Nuclear translocation of NF-κB is regulated from the cytoplasm by IκB kinase-β (IKKβ), which earmarks inhibitors of NF-κB for polyubiquination and proteasome-mediated degradation. Activation of IKKβ is contingent upon signal-induced phosphorylation of its T loop at Ser-177/Ser-181. T loop phosphorylation also renders IKKβ a substrate for monoubiquitination in cells exposed to chronic activating cues, such as the Tax oncoprotein or sustained signaling through proinflammatory cytokine receptors. Here we provide evidence that the T loop-proximal residue Lys-163 in IKKβ serves as a major site for signal-induced monoubiquitination with significant regulatory potential. Conservative replacement of Lys-163 with Arg yielded a monoubiquitination-defective mutant of IKKβ that retains kinase activity in Tax-expressing cells but is impaired for activation mediated by chronic signaling from the type 1 receptor for tumor necrosis factor-α. Phosphopeptide mapping experiments revealed that the Lys-163 → Arg mutation also interferes with proper in vivo but not in vitro phosphorylation of cytokine-responsive serine residues located in the distal C-terminal region of IKKβ. Taken together, these data indicate that chronic phosphorylation of IKKβ at Ser-177/Ser-181 leads to monoubiquitin attachment at nearby Lys-163, which in turn modulates the phosphorylation status of IKKβ at select C-terminal serines. This mechanism for post-translational cross-talk may play an important role in the control of IKKβ signaling during chronic inflammation.