The hypoglycemic effects of high dose salicylates in the treatment of diabetes were documented before the advent of insulin. However, the molecular mechanisms by which salicylates exert these anti-diabetic effects are not well understood. In this study, we analyzed the effects of aspirin (acetylsalicylic acid) on serine phosphorylation of insulin receptor substrate I (IRS-1) in cells treated with tumor necrosis factor (TNF)-α. Phosphorylation of IRS-1 at Ser307, Ser267, and Ser612 was monitored by immunoblotting with phospho-specific IRS-1 anti-bodies. In 3T3-L1 and Hep G2 cells, phosphorylation of IRS-1 at Ser307 in response to TNF-α treatment correlated with phosphorylation of JNK, c-Jun, and degradation of IκBα. Moreover, phosphorylation of IRS-1 at Ser 307 in embryo fibroblasts derived from either JNK or IKK knockout mice was reduced when compared with that in the wild-type controls. Taken together, these data suggest that serine phosphorylation of IRS-1 in response to TNF-α is mediated, in part, by JNK and IKK. Interestingly, aspirin treatment inhibited the phosphorylation of IRS-1 at Ser307 as well as the phosphorylation of JNK, c-Jun, and degradation of IκBα. Furthermore, other serine kinases including Akt, extracellular regulated kinase, mammalian target of rapamycin, and PKCζ were also activated by TNF-α (as assessed by phospho-specific antibodies). Phosphorylation of IRS-1 at Ser267 and Ser612 correlated with the activation of these kinases. Phosphorylation of Akt and the mammalian target of rapamycin (but not extracellular regulated kinase or PKCζ) in response to TNF-α was inhibited by aspirin treatment. Finally, aspirin rescued insulin-induced glucose uptake in 3T3-L1 adipocytes pretreated with TNF-α. We conclude that aspirin may enhance insulin sensitivity by protecting IRS proteins from serine phosphorylation catalyzed by multiple kinases.