To examine the molecular mechanisms by which plasma amino acid elevation impairs insulin action, we studied seven healthy men twice in random order during infusion of an amino acid mixture or saline (total plasma amino acid ∼6 vs. ∼2 mmol/l). Somatostatin-insulin-glucose clamps created conditions of low peripheral hyperinsulinemia (∼100 pmol/l, 0–180 min) and prandial-like peripheral hyperinsulinemia (∼430 pmol/l, 180–360 min). At low peripheral hyperinsulinemia, endogenous glucose production (EGP) did not change during amino acid infusion but decreased by ∼70% during saline infusion (EGP_{150–180 min} 11 ± 1 vs. 3 ± 1 μmol · kg⁻¹ · min⁻¹, P = 0.001). Prandial-like peripheral hyperinsulinemia completely suppressed EGP during both protocols, whereas whole-body rate of glucose disappearance (R_d) was ∼33% lower during amino acid infusion (R_{d 330–360 min} 50 ± 4 vs. 75 ± 6 μmol · kg⁻¹ · min⁻¹, P = 0.002) indicating insulin resistance. In skeletal muscle biopsies taken before and after prandial-like peripheral hyperinsulinemia, plasma amino acid elevation markedly increased the ability of insulin to activate S6 kinase 1 compared with saline infusion (∼3.7- vs. ∼1.9-fold over baseline). Furthermore, amino acid infusion increased the inhibitory insulin receptor substrate-1 phosphorylation at Ser312 and Ser636/639 and decreased insulin-induced phosphoinositide 3-kinase activity. However, plasma amino acid elevation failed to reduce insulin-induced Akt/protein kinase B and glycogen synthase kinase 3α phosphorylation. In conclusion, amino acids impair 1) insulin-mediated suppression of glucose production and 2) insulin-stimulated glucose disposal in skeletal muscle. Our results suggest that overactivation of the mammalian target of rapamycin/S6 kinase 1 pathway and inhibitory serine phosphorylation of insulin receptor substrate-1 underlie the impairment of insulin action in amino acid–infused humans.