Derangements in skeletal muscle fatty acid (FA) metabolism associated with insulin resistance in obesity appear to involve decreased FA oxidation and increased accumulation of lipids such as ceramides and diacylglycerol (DAG). We investigated potential lipid-related mechanisms of metformin (Met) and/or exercise (Ex) for blunting the progression of hyperglycemia/hyperinsulinemia and skeletal muscle insulin resistance in female Zucker diabetic fatty rats (ZDF), a high-fat (HF) diet-induced model of diabetes. Lean and ZDF rats consumed control (C), or HF diet (HF, 48 kcal% fat) alone or with Met (500mg/kg, HF-Met), with treadmill Ex (HF-Ex), or with both Ex and Met (HF-E+M) interventions for 8 weeks. HF-fed ZDF rats developed hyperglycemia (mean: 24.4+/-2.1mM), impairments in muscle insulin-stimulated glucose transport, increases in the FA transporter FAT/CD36, and increases in total ceramide and DAG content. The development of hyperglycemia was significantly attenuated with all interventions, as was skeletal muscle FAT/CD36 abundance, ceramide and DAG content. Interestingly, improvements in insulin-stimulated glucose transport and increased GLUT4 transporter expression in isolated muscle were seen only in conditions which included exercise training. Reduced FA oxidation and increased triacylglycerol synthesis in isolated muscle were observed with all ZDF rats compared to Lean (P < 0.01), and were unaltered by therapeutic intervention. However, exercise did induce modest increases in peroxisome proliferator-activated receptor-? coactivator-1?, citrate synthase and ?-hydroxyacyl-CoA dehydrogenase activity. Thus, reduction of skeletal muscle FAT/CD36, and content of ceramide and DAG may be important mechanisms by which exercise training blunts the progression of diet-induced insulin resistance in skeletal muscle.