High fat (HF) diets can induce insulin resistance (IR) by altering skeletal muscle lipid metabolism. An imbalance between fatty acid (FA) uptake and oxidation results in intramuscular lipid accumulation, which can impair the insulin-signaling cascade. Adiponectin (Ad) is an insulin sensitizing adipokine known to stimulate skeletal muscle FA oxidation and reduce lipid accumulation. Evidence of Ad resistance has been shown in obesity and following chronic HF feeding, and may contribute to lipid accumulation observed in these conditions. Whether Ad resistance precedes and is associated with the development of IR is unknown. We conducted a time course HF feeding trial for 3 days, 2 weeks or 4 weeks to determine the onset of Ad resistance and identify the ensuing changes in lipid metabolism and insulin signaling leading to IR in skeletal muscle. Adiponectin stimulated FA oxidation (+28%, p0.05) and ACC phosphorylation (+34%, p0.05) in control animals, but failed to do so in any HF fed group (i.e. as early as 3 days). By 2 weeks, plasma membrane FA transporters and intramuscular diacylglycerol (DAG) and ceramide were increased and insulin-stimulated phosphorylation of both Akt and AS160 was blunted compared to control animals. After 4 weeks of HF feeding, maximal insulin stimulated glucose transport was impaired compared to control. Taken together, our results demonstrate that an early loss of Ad's stimulatory effect on FA oxidation precedes an increase in plasmalemmal FA transporters and the accumulation of intramuscular DAG and ceramide, blunted insulin signaling and ultimately impaired maximal insulin stimulated glucose transport into skeletal muscle induced by HF diets.