Reduced expression of the NAD+-dependent deacetylase, SIRT3, has been associated with insulin resistance and metabolic dysfunction in humans and rodents. In this study we investigated whether specific overexpression of SIRT3 in vivo in skeletal muscle could prevent HFD-induced muscle insulin resistance. To address this we used a muscle-specific adeno-associated virus (AAV) to overexpress SIRT3 in rat tibialis and EDL muscles. Mitochondrial substrate oxidation, substrate switching and oxidative enzyme activity were assessed in skeletal muscle with and without SIRT3 overexpression. Muscle-specific insulin action was also assessed by hyperinsulinaemic-euglycaemic clamps in rats that underwent a 4-week HFD-feeding protocol. Ex vivo functional assays revealed elevated activity of selected SIRT3-target enzymes including hexokinase, isocitrate dehydrogenase and pyruvate dehydrogenase that was associated with an increase in the ability to switch between fatty acid and glucose-derived substrates in muscle with SIRT3 overexpression. However, during the clamp, muscle from rats fed a HFD with increased SIRT3 expression displayed equally impaired glucose uptake and insulin-stimulated glycogen synthesis as the contralateral control muscle. Intramuscular triglyceride content was similarly increased in muscle of high fat fed rats, regardless of SIRT3 status. Thus, despite SIRT3 KO mouse models indicating many beneficial metabolic roles for SIRT3, our findings show that muscle-specific overexpression of SIRT3 has only minor effects on the acute development of skeletal muscle insulin resistance in high fat fed rats.