Although evidence has been accumulating that type 2 diabetes mellitus (T2DM) is accompanied by mitochondrial dysfunction in skeletal muscle, a causal link between mitochondrial dysfunction and the pathogenesis of the disease remains unclear. Our study focuses on an early stage of the disease in order to determine whether mitochondrial dysfunction contributes to the development of T2DM. The fructose-fed (FF) rat was used as an animal model of early T2DM. Mitochondrial respiration and acylcarnitine species were measured in oxidative (soleus) and glycolytic (extensor digitorum longus, EDL) muscle. Although FF rats displayed characteristic signs of T2DM, including hyperglycemia, hyperinsulinemia, hypertriglyceridemia, mitochondrial content was preserved in both muscles from FF rats. The EDL muscle had reduced complex I and complex I+II respiration in the presence of pyruvate, but not glutamate. The decrease in pyruvate-supported respiration was due to a decrease in pyruvate dehydrogenase activity. Accumulation of C14:1 and C14:2 acylcarnitine species and a decrease in respiration supported by long-chain acylcarnitines but not acetylcarnitine indicated dysfunctional β-oxidation in the EDL muscle. In contrast, the soleus muscle showed preserved mitochondrial respiration, pyruvate dehydrogenase activity and increased fatty acid oxidation as evidenced by overall reduced acylcarnitine levels. Aconitase activity, a sensitive index of reactive oxygen species production in mitochondria, was exclusively reduced in EDL muscle, which showed lower levels of the antioxidant enzymes thioredoxin reductase and glutathione peroxidase. We here show that the glycolytic EDL muscle is more prone to an imbalance between energy supply and oxidation caused by insulin resistance than the oxidative soleus muscle.