Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47(phox) is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47(phox). We show that p47(phox) is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47(phox) not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47(phox) is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47(phox) was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47(phox) activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47(phox) and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr(133), Ser(288) and Thr(356), targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47(phox) after LPS stimulation. We conclude that IRAK-4 phosphorylates p47(phox) and regulates NADPH oxidase activation after LPS stimulation.