Suppressor of cytokine signaling (SOCS) proteins and/or activation of the pro-inflammatory pathway have been postulated as possible mechanisms that may contribute to skeletal muscle insulin resistance. Thus, the aims of the present investigation were to determine in high-fat fed skeletal muscle: 1) if SOCS-3 protein concentration is increased, 2) whether co-immunoprecipitation of SOCS-3 with the insulin receptor- subunit and/or IRS-1 is increased, and 3) if select components of the pro-inflammatory pathway are altered. Thirty-two male Sprague-Dawley rats were assigned to either Control (Con, n=16) or High-fat Fed (HF, n=16) dietary groups for 12 wks and then subjected to hind limb perfusions in the presence (n=8/group) or absence (n=8/group) of insulin. Insulin-stimulated skeletal muscle 3-MG transport rates and PI-3 kinase activity were greater (p<0.05) in Con. IRS-1 tyrosine phosphorylation was decreased (p<0.05) and IRS-1 serine 307 phosphorylation was increased (p<0.05) in HF. Insulin receptor- (IR-) subunit co-immunoprecipitation with IRS-1 was reduced in HF. SOCS-3 protein concentration, and SOCS-3 co-immunoprecipitation with both the IR- subunit and IRS-1 was increased (p<0.05) in HF. IKK/ serine phosphorylation was increased (p<0.05), IB protein concentration was decreased (p<0.05) and IB serine phosphorylation was increased (p<0.05) in HF. Increased co-localization of SOCS-3 with both the IR- subunit and IRS-1 may provide steric hindrance that prevents IRS-1 from interacting with IR-, while increased IKK serine phosphorylation may contribute to increasing IRS-1 serine phosphorylation, both of which independently can have deleterious effects on insulin-stimulated PI-3 kinase activation in high-fat fed rodent skeletal muscle.