Dietary factors have been implicated in the development of hepatic and peripheral insulin resistance. Differently composed diets can induce insulin resistance in different ways, but the mechanisms underlying these phenomena are not yet clear. This study was conducted to evaluate whether dietary composition change affects insulin resistance in the skeletal muscles of rats fed high-carbohydrate diet or high-fat diet. We assessed glucose transport in the skeletal muscles of rats in vitro. Diets given were rat chow, high-starch (HT), high-sucrose (HS), high-fat high-starch (HFHT), high-fat high-sucrose (HFHS, HF), HF with fish oil (HF+FO), and HF with linseed oil (HF+LO). Both of HS diet and HT diet with or without high-fat depressed insulin-stimulated glucose transport compared with chow diet, but there were no significant difference between groups. HF diet markedly decreased the insulin-stimulated glucose transport, and fish oil improved this partially, but linseed oil did not significantly. Percent visceral fat pad mass, plasma insulin and triglyceride in high carbohydrate or high-fat diet groups that developed muscle insulin resistance were much higher compared with chow diet group. Fish oil and linseed oil decreased percent visceral fat pad mass, and fish oil decreased plasma insulin and triglyceride. The composition of fat diet was more important factor than that of carbohydrate diet to induce muscle insulin resistance assessed by glucose transport in vitro. Plasma triglyceride and insulin concentrations seemed to be important factors to induce muscle insulin resistance in rats. [J Physiol Sci. 2006;56 Suppl:S17]