Omega-3 polyunsaturated fatty acids (n-3 PUFAs) enhance insulin sensitivity and glucose homeostasis in rodent models of insulin resistance. These beneficial effects have been linked with anti-inflammatory properties, but emerging data suggests that the mechanisms may also converge on mitochondria. We evaluated the influence of dietary n-3 PUFAs on mitochondrial physiology and muscle lipid metabolites in the context of high-fat diet (HFD) in mice. Mice were fed control diets (10% fat), HFD (60% fat) or HFD with fish oil (HFD+FO, 3.4% kcals from n-3 PUFAs) for 10 weeks. Body mass and fat mass increased similarly in HFD and HFD+FO, but n-3 PUFAs attenuated the glucose intolerance that developed with HFD and increased expression of genes that regulate glucose metabolism in skeletal muscle. Despite similar muscle triglyceride levels in HFD and HFD+FO, long chain acyl coenzyme-As and ceramides were lower in the presence of fish oil. Mitochondrial abundance and oxidative capacity were similarly increased in HFD and HFD+FO compared to controls. Hydrogen peroxide production was similarly elevated in HFD and HFD+FO in isolated mitochondria, but not in permeabilized muscle fibers, likely due to increased activity and expression of catalase. These results support a hypothesis that n-3 PUFAs protect glucose tolerance, in part, by preventing the accumulation of bioactive lipid mediators that interfere with insulin action. Furthermore, the respiratory function of skeletal muscle mitochondria does not appear to be a major factor in sphingolipid accumulation, glucose intolerance, or the protective effects of n-3 PUFAs.