Abstract Tumor necrosis factor-α (TNFα) is a multifunctional cytokine involved in the pathophysiology of many chronic inflammatory diseases. TNFα activation of the nuclear factor κB (NFκB) signaling pathway particularly in macrophages has been implicated in many diseases. We demonstrate here that G-protein coupled receptor kinase-2 and 5 (GRK2 and 5) regulate TNFα-induced NFκB signaling in macrophages. RNAi knockdown of GRK2 or 5 in macrophages significantly inhibited TNFα-induced IκBα phosphorylation and degradation, NFκB activation, and expression of the NFκB-regulated gene macrophage inflammatory protein-1β. Consistent with these results, over-expression of catalytically active GRK2 or 5 enhanced TNFα-induced NFκB activity. In addition, we show that GRK2 and 5 interact with IκBα in intact cells as well as in vitro via the N-terminal domain of IκBα and that IκBα is a substrate for GRK2 and 5 in vitro. Taken together, these results demonstrate that GRK2 and 5 are important mediators of TNFα-induced NFκB activity and suggest that GRKs could be potential therapeutic targets for treating TNFα-mediated inflammatory diseases.