In spite of its central roles in cell cycle progression, senescence, and aging, knowledge about the posttranslational regulation of P16 (also known as INK4A and MTS1) remains limited. While it has been reported that P16 could be phosphorylated at Ser7, Ser8, Ser140, and Ser152, the corresponding kinases have not been identified yet. Here we report that IKKbeta, a primary kinase for IkappaBalpha phosphorylation, is involved in P16 phosphorylation. Immunoprecipitation and kinase assays showed that IKKbeta specifically binds to P16 and phosphorylates P16 at Ser8 in WI38 cells. Biochemical characterization of phosphomimetic Ser-->Glu P16 mutants demonstrated that phosphorylation at Ser8 of P16 brings about a significant loss of its cyclin-dependent kinase (CDK) 4-inhibitory activity while P16 retains structurally and functionally intact upon phosphorylation at Ser7, Ser140, and Ser152. Our results reveal the novel role of IKKbeta in P16 phosphorylation and broaden our understanding of the regulation of P16.