Most lifestyle-related chronic diseases are characterized by low-grade systemic inflammation and insulin resistance. Excessive tumor necrosis factor-α (TNF-α) concentrations have been implicated in the development of insulin resistance, but direct evidence in humans is lacking. Here, we demonstrate that TNF-α infusion in healthy humans induces insulin resistance in skeletal muscle, without effect on endogenous glucose production, as estimated by a combined euglycemic insulin clamp and stable isotope tracer method. TNF-α directly impairs glucose uptake and metabolism by altering insulin signal transduction. TNF-α infusion increases phosphorylation of p70 S6 kinase, extracellular signal-regulated kinase-1/2, and c-Jun NH 2-terminal kinase, concomitant with increased serine and reduced tyrosine phosphorylation of insulin receptor substrate-1. These signaling effects are associated with impaired phosphorylation of Akt substrate 160, the most proximal step identified in the canonical insulin signaling cascade regulating GLUT4 translocation and glucose uptake. Thus, excessive concentrations of TNF-α negatively regulate insulin signaling and whole-body glucose uptake in humans. Our results provide a molecular link between low-grade systemic inflammation and the metabolic syndrome.