Dietary intervention for preventing postprandial increases in glucose level by replacing high-glycemic index (GI) carbohydrates with lower-GI carbohydrate has been proposed as a strategy for treating insulin-resistant metabolic disorders such as type II diabetes. In this study, we examined the effect of short-term replacement of starch with a low-GI disaccharide, isomaltulose, on insulin action in skeletal muscle. Male Wistar rats were fed isomaltulose for 12 h during their dark cycle. In isolated epitrochlearis muscle, insulin-induced glucose uptake was greater in tissue from rats treated with isomaltulose than from those treated with starch. This insulin-sensitizing effect occurred independently of changes visceral fat mass. To determine whether this sensitization was specific to insulin stimulation, we also measured glucose uptake in response to exercise. In isolated epitrochlearis muscles from rats that performed swimming exercise, exercise-induced glucose uptake was higher in isomaltulose-treated than starch-treated animals. This amplification was associated with increased phosphorylation of exercise-induced AMP-activated protein kinase. In conclusion, our results demonstrate that short-term replacement of starch with isomaltulose enhances both insulin-dependent and -independent glucose uptake in isolated skeletal muscle. This transient replacement of carbohydrate with isomaltulose, together with exercise, represents a potentially effective approach for the management of insulin resistance.