Abundant evidence supports a crucial role for dietary factors in the induction and maintenance of insulin resistance. At the cellular and tissue level, the availability of substrates for cellular energy production may play an important role in metabolic regulation and, in particular, in determining the response to insulin stimulation. The infusion of amino acids or fatty acids decreases insulin-stimulated glucose disposal in vivo; sustained hyperglycemia also induces insulin resistance. To determine whether nutrients directly affect insulin signaling, we have evaluated the impact of fatty acids, amino acids, and activation of the hexosamine pathway on insulin signaling in both cultured cells and animal models. We demonstrate that fatty acids and amino acids inhibit early post-receptor steps in insulin action, including tyrosine phosphorylation of insulin receptor substrate (IRS) proteins and activation of phosphatidylinositol 3-kinase (PI3-kinase), both in vitro and in several in vivo models. Similarly, activation of the hexosamine pathway by infusion of glucosamine also reduces insulin-stimulated phosphorylation of IRS proteins, activation of PI3-kinase, and activation of glycogen synthase. These data suggest that nutrients directly modulate insulin signaling, perhaps via common pathways, and thus contribute to cellular insulin resistance.