TNFα (tumour necrosis factor α) is a multifunctional cytokine involved in the pathophysiology of many chronic inflammatory diseases. TNFα activation of the NF-κB (nuclear factor κB) signalling pathway particularly in macrophages has been implicated in many diseases. We demonstrate in the present study that GRK2 and GRK5 (G-protein-coupled-receptor kinases 2 and 5) regulate TNFα-induced NF-κB signalling in Raw 264.7 macrophages. RNAi (RNA interference) knockdown of GRK2 or GRK5 in macrophages significantly inhibited TNFα-induced IκBα (inhibitory κBα) phosphorylation and degradation, NF-κB activation and expression of the NF-κB-regulated gene MIP1β (macrophage inflammatory protein 1β). Consistent with these results, overexpression of GRK2 or GRK5 enhanced TNFα-induced NF-κB activity. In addition, we show that GRK2 and GRK5 interacted with IκBα via the N-terminal domain of IκBα and that IκBα is a substrate for GRK2 and GRK5 in vitro. Furthermore, we also found that GRK5, but not GRK2, phosphorylated IκBα at the same amino acid residues (Ser³²/Ser³⁶) as that of IKKβ (IκB kinase β). Interestingly, associated with these results, knockdown of IKKβ in Raw 264.7 macrophages did not affect TNFα-induced IκBα phosphorylation. Taken together, these results demonstrate that both GRK2 and GRK5 are important and novel mediators of a non-traditional IκBα/NF-κB signalling pathway.