We determined whether chronic endothelin-1 (ET-1) treatment could lead to in vivo insulin resistance. Like insulin, ET-1 acutely stimulated glucose transport in isolated soleus muscle strips of WKY rats. ET-1 pretreatment (1 h) decreased insulin-stimulated glucose transport in muscle strips (−23%). Both ET-1-mediated effects were generated through ET_A receptors, because a specific ET_A receptor antagonist (BQ610) blocked these effects of ET-1. Osmotic minipumps were used to treat normal rats with ET-1 for 5 days. Subsequent hyperinsulinemic-euglycemic clamps showed that ET-1 treatment led to an ∼30% decrease in insulin-stimulated glucose disposal rates in male and female rats. In addition, ex vivo study of soleus muscle strips showed decreased glucose transport into muscle from ET-1-treated animals. With respect to insulin signaling, chronic in vivo ET-1 treatment led to a 30–40% decrease in IRS-I protein content, IRS-I-associated p110_α, and AKT activation. In summary, 1) in vitro ET-1 pretreatment leads to decreased insulin-stimulated glucose transport in skeletal muscle strips; 2) chronic ET-1 administration in vivo leads to whole-body insulin resistance, with decreased skeletal muscle glucose transport and impaired insulin signaling; and 3) elevated ET-1 levels may be a cause of insulin resistance in certain pathophysiologic states.