Plasma concentrations of amino acids are frequently elevated in insulin-resistant states, and a protein-enriched diet can impair glucose metabolism. This study examined effects of short-term plasma amino acid (AA) elevation on whole-body glucose disposal and cellular insulin action in skeletal muscle. Seven healthy men were studied for 5.5 h during euglycemic (5.5 mmol/l), hyperinsulinemic (430 pmol/l), fasting glucagon (65 ng/l), and growth hormone (0.4 μg/l) somatostatin clamp tests in the presence of low (∼1.6 mmol/l) and increased (∼4.6 mmol/l) plasma AA concentrations. Glucose turnover was measured with d-[6,6-²H₂]glucose. Intramuscular concentrations of glycogen and glucose-6-phosphate (G6P) were monitored using ¹³C and ³¹P nuclear magnetic resonance spectroscopy, respectively. A ∼2.1-fold elevation of plasma AAs reduced whole-body glucose disposal by 25% (P < 0.01). Rates of muscle glycogen synthesis decreased by 64% (180–315 min, 24 ± 3; control, 67 ± 10 μmol · l⁻¹ · min⁻¹; P < 0.01), which was accompanied by a reduction in G6P starting at 130 min (ΔG6P_{260–300 min}, 18 ± 19; control, 103 ± 33 μmol/l; P < 0.05). In conclusion, plasma amino acid elevation induces skeletal muscle insulin resistance in humans by inhibition of glucose transport/phosphorylation, resulting in marked reduction of glycogen synthesis.