Mitochondrial dysfunction in skeletal muscle is cause of impaired lipid oxidation and accumulation of intramyocellular lipid (IMCL) in type 2 diabetes and insulin resistance syndrome. 3,5-diiodo-L-thyronine (T2), when administered to high fat diet (HFD)-fed rats, leads to: reduction in body adiposity, increase in hepatic fatty acid oxidation, and improved metabolic parameters and glucose tolerance, by preventing IMCL accumulation and insulin resistance, without deleterious side effects. We investigated the in vivo effects of 4-weeks administration of T2 to HFD rats on the skeletal muscle metabolic/structural phenotype with a focus on mitochondrial protein profile and function by two-dimensional gel electrophoreses (2D-E) and Blue-Native (BN) PAGE. T2 produces an increase in fast/glycolytic muscle fibres and, at the mitochondrial level, counteracts several HFD-induced changes. BN-PAGE revealed a modified profile of individual complexes in HFD mitochondria vs normal ones, this pattern being normalized in mitochondria from T2-treated animals with a concomitant enhancement of the activity of complex I and IV and a normalization of that of complex V. These findings suggest that while the slow-oxidative HFD-induced phenotype associates with a perturbed protein expression pattern, affecting glucose, fatty acid, the contractile apparatus, and the respiratory chain, T2, on the other hand, prevents such alterations and, in some cases, produces, opposite beneficial effects.