The aims of this investigation were to evaluate the effect of an amino acid supplement on the glucose response to an oral glucose challenge (experiment 1) and to evaluate whether differences in blood glucose response were associated with increased skeletal muscle glucose uptake (experimental 2). Experiment 1 rats were gavaged with either glucose (CHO), glucose plus an amino acid mixture (CHO-AA-1), glucose plus an amino acid mixture with increased leucine concentration (CHO-AA-2), or water (PLA). CHO-AA-1 and CHO-AA-2 had reduced blood glucose responses compared with CHO, with no difference in insulin among these treatments. Experiment 2 rats were gavaged with either CHO or CHO-AA-1. Fifteen minutes after gavage, a bolus containing 2-[(3)H]deoxyglucose and [U-(14)C]mannitol was infused via a tail vein. Blood glucose was significantly lower in CHO-AA-1 than in CHO, whereas insulin responses were similar. Muscle glucose uptake was higher in CHO-AA-1 compared with CHO in both fast-twitch red (8.36 ± 1.3 vs. 5.27 ± 0.7 μmol·g(-1)·h(-1)) and white muscle (1.85 ± 0.3 vs. 1.11 ± 0.2 μmol·g(-1)·h(-1)). There was no difference in Akt/PKB phosphorylation between treatment groups; however, the amino acid treatment resulted in increased AS160 phosphorylation in both muscle fiber types. Glycogen synthase phosphorylation was reduced in fast-twitch red muscle of CHO-AA-1 compared with CHO, whereas mTOR phosphorylation was increased. These differences were not noted in fast-twitch white muscle. These findings suggest that amino acid supplementation can improve glucose tolerance by increasing skeletal muscle glucose uptake and intracellular disposal through enhanced intracellular signaling.