Inflammation contributes to insulin resistance in diabetes and obesity. Mouse Pelle-like kinase (mPLK, homolog of human IL-1 receptor-associated kinase (IRAK)) participates in inflammatory signaling. We evaluated IRS-1 as a novel substrate for mPLK that may contribute to linking inflammation with insulin resistance. Wild-type mPLK, but not a kinase-inactive mutant (mPLK-KD), directly phosphorylated full-length IRS-1 in vitro. This in vitro phosphorylation was increased when mPLK was immunoprecipitated from tumor necrosis factor (TNF)-α-treated cells. In NIH-3T3IR cells, wild-type mPLK (but not mPLK-KD) co-immunoprecipitated with IRS-1. This association was increased by treatment of cells with TNF-α. Using mass spectrometry, we identified Ser24 in the pleckstrin homology (PH) domain of IRS-I as a specific phosphorylation site for mPLK, IRS-1 mutants S24D or S24E (mimicking phosphorylation at Ser24) had impaired ability to associate with insulin receptors resulting in diminished tyrosine phosphorylation of IRS-1 and impaired ability of IRS-1 to bind and activate PI-3 kinase in response to insulin. IRS-1-S24D also had an impaired ability to mediate insulin-stimulated translocation of GLUT4 in rat adipose cells. Importantly, endogenous mPLK/IRAK was activated in response to TNF-α or interleukin 1 treatment of primary adipose cells. In addition, using a phospho-specific antibody against IRS-1 phosphorylated at Ser24, we found that interleukin-1 or TNF-α treatment of Fao cells stimulated increased phosphorylation of endogenous IRS-1 at Ser24. We conclude that IRS-1 is a novel physiological substrate for mPLK. TNF-α-regulated phosphorylation at Ser24 in the pleckstrin homology domain of IRS-1 by mPLK/IRAK represents an additional mechanism for cross-talk between inflammatory signaling and insulin signaling that may contribute to metabolic insulin resistance.