High-fat (HF) diets induce insulin resistance and alter lipid metabolism, although controversy exists regarding the impact of saturated vs. polyunsaturated fats. Adiponectin (Ad) stimulates fatty acid (FA) oxidation and improves insulin sensitivity in humans and rodents, due in part to the activation of AMP-activated protein kinase (AMPK) and subsequent deactivation of acetyl coenzyme-A carboxylase (ACC). In genetically obese diabetic mice, this acute stimulatory effect on AMPK in muscle is lost. The ability of a HF diet to induce skeletal muscle Ad resistance has not been examined. The purpose of this study was to determine whether Ads effects on FA oxidation and AMPK/ACC would be reduced following different HF diets, and if this coincided with the development of impaired maximal insulin stimulated glucose transport. Rats were fed a control (10% kcal fat, CON), high-unsaturated fat (60% kcal safflower oil, SAFF) or high-saturated fat diet (60% kcal lard, LARD) for 4 weeks. Following the dietary intervention, glucose transport, lipid metabolism and AMPK/ACC phosphorylation were measured in the presence and absence of globular Ad (gAd, 2.5?g/ml) in isolated soleus muscle. LARD rats showed reduced rates of maximal insulin-stimulated glucose transport compared to CON and SAFF (+68%, vs. +172%, +184% p?0.001). gAd increased pACC (+25%, p?0.01) and FA oxidation (+28%, p?0.05) in CON rats, but not in either HF group. Thus, 4 weeks of HF feeding results in the loss of globular Ads stimulatory effect on ACC phosphorylation and muscle FA oxidation, and this can occur independently of impaired maximal insulin-stimulated glucose transport.