Increased lipid metabolism in muscle is associated with insulin resistance and therefore, many strategies have been employed to alter fatty acid metabolism and study the impact on insulin action. Metabolism of fatty acid requires activation to fatty acyl CoA by Acyl CoA synthases (ACSL) and fatty acyl CoA can be hydrolysed by Acyl CoA thioesterases (Acot). Thioesterase activity is low in muscle, so we overexpressed Acot7 in muscle of chow and high-fat diet (HFD) rats and investigated effects on insulin action. Acot7 overexpression modified specific phosphatidylcholine and phosphatidylethanolamine species in tibialis muscle of chow rats to levels similar to those observed in control HFD muscle. The changes in phospholipid species did not alter glucose uptake in tibialis muscle under hyperinsulinaemic/euglycaemic clamped conditions. Acot7 overexpression in white extensor digitorum longus (EDL) muscle increased complete fatty acid oxidation ex-vivo but was not associated with any changes in glucose uptake in-vivo, however overexpression of Acot7 in red EDL reduced insulin-stimulated glucose uptake in-vivo which correlated with increased incomplete fatty acid oxidation ex-vivo. In summary, although overexpression of Acot7 in muscle altered some aspects of lipid profile and metabolism in muscle, this had no major effect on insulin-stimulated glucose uptake.