The primary purpose of this study was to determine the effect of prior exercise on insulin-stimulated glucose uptake with physiologic insulin in isolated muscles of mice. Male C57BL/6 mice completed a 60min treadmill exercise protocol or were sedentary. Paired epitochlearis, soleus, and extensor digitorum longus (EDL) muscles were incubated with [ ³H]-2-deoxyglucose without or with insulin (60µU/ml) to measure glucose uptake. Insulin-stimulated glucose uptake for paired muscles was calculated by subtracting glucose uptake without insulin from glucose uptake with insulin. Muscles from other mice were assessed for glycogen and AMPK Thr¹⁷² phosphorylation. Exercised versus sedentary mice had decreased glycogen in epitrochlearis (48%, P<0.001), soleus (51%, P<0.001) and EDL (41%, P<0.01) and increased AMPK Thr¹⁷² phosphorylation (P<0.05) in epitrochlearis (1.7 fold), soleus (2.0 fold) and EDL (1.4 fold). Insulin-independent glucose uptake was increased 30min post-exercise versus sedentary in the epitrochlearis (1.2 fold, P<0.001), soleus (1.4 fold, P<0.05) and EDL (1.3 fold, P<0.01). Insulin-stimulated glucose uptake was increased (P<0.05) ~85min after exercise in the epitrochlearis (sedentary: 0.266 ± 0.045 mmol x g^-1 x 15min^-1; exercised: 0.414 ± 0.051) and soleus (sedentary: 0.102 ± 0.049; exercised: 0.347 ± 0.098) but not in the EDL. Akt Ser⁴⁷³ and Akt Thr³⁰⁸ phosphorylation for insulin-stimulated muscles did not differ in exercised versus sedentary. These results demonstrate enhanced submaximal insulin-stimulated glucose uptake in the epitrochlearis and soleus of mice 85min post-exercise and suggest that it will be feasible to probe the mechanism of enhanced post-exercise insulin sensitivity using genetically modified mice.