Mitochondrial dysfunction in muscle has been implicated to play a causative role or being an indirect consequence of insulin resistance in type-2 diabetes. In order to investigate potential diabetes-related alterations in the mitochondrial proteome of muscle, we have carried out a mass spectrometry-based proteomic analysis of the gastrocnemius muscle from normal versus diabetic Goto-Kakizaki rats. A generally perturbed protein expression pattern was observed in the mitochondria-enriched fraction from diabetic muscle. Various mitochondrial markers, including NADH dehydrogenase, cytochrome b-c1 complex and isocitrate dehydrogenase were reduced in diabetic preparations. Isoforms of pyruvate dehydrogenase and ATP synthase exhibited differential changes in their abundance. The altered protein expression levels of these key metabolic enzymes might trigger a diabetes-dependent decrease in mitochondrial oxidative phosphorylation levels. The proteomic findings presented here support the idea that mitochondrial abnormalities are involved in the molecular pathogenesis of type-2 diabetes and may be crucial for the development of insulin resistance.