Mitochondrial dysfunction in skeletal muscle has been implicated in the development of insulin resistance and type 2 diabetes. Considering the importance of mitochondrial dynamics in mitochondrial and cellular functions, we hypothesized that obesity and excess energy intake shift the balance of mitochondrial dynamics, further contributing to mitochondrial dysfunction and metabolic deterioration in skeletal muscle. First, we revealed that excess palmitate (PA), but not hyperglycemia, hyperinsulinemia, or elevated tumor necrosis factor alpha, induced mitochondrial fragmentation and increased mitochondrion-associated Drp1 and Fis1 in differentiated C2C12 muscle cells. This fragmentation was associated with increased oxidative stress, mitochondrial depolarization, loss of ATP production, and reduced insulin-stimulated glucose uptake. Both genetic and pharmacological inhibition of Drp1 attenuated PA-induced mitochondrial fragmentation, mitochondrial depolarization, and insulin resistance in C2C12 cells. Furthermore, we found smaller and shorter mitochondria and increased mitochondrial fission machinery in the skeletal muscle of mice with genetic obesity and those with diet-induced obesity. Inhibition of mitochondrial fission improved the muscle insulin signaling and systemic insulin sensitivity of obese mice. Our findings indicated that aberrant mitochondrial fission is causally associated with mitochondrial dysfunction and insulin resistance in skeletal muscle. Thus, disruption of mitochondrial dynamics may underlie the pathogenesis of muscle insulin resistance in obesity and type 2 diabetes.T he prevalence of obesity and type 2 diabetes is increasing at an alarming rate in industrialized countries, partly due to excess food intake and physical inactivity. Excess dietary fat and sugar leads to increased flux of energy fuel substrates and increased lipid burden in peripheral tissues. Skeletal muscle is the major site of glucose uptake and metabolism. Increased fatty acid (FA) uptake contributes to increased lipid accumulation in skeletal muscle, leading to lipotoxicity, which is known to impair muscle insulin sensitivity (2,20). In addition, the intracellular lipid metabolites have been shown to activate serine/threonine protein kinases and suppress insulin actions (37).Mitochondria are important organelles for cellular function through regulation of energy metabolism, ATP generation, and calcium handling. Substantial evidence shows that mitochondrial dysfunction and impairment of the oxidative capacity in skeletal muscle are key mechanisms mediating insulin resistance (24, 34). A reduction in the number and function of mitochondria has been documented in the skeletal muscle of type 2 diabetic patients and animals. For example, the activity of the electron transport chain in subsarcolemmal mitochondria is dramatically reduced in type 2 diabetic and obese subjects, compared with that in lean subjects (36). Furthermore, patients with severe insulin resistance exhibit decreased mitochondrial oxidative activity and ATP s...