Peptide YY3-36 (PYY3-36) is released by the gut in response to nutrient ingestion. It modulates the activities of orexigenic neuropeptide Y (NPY) neurons and anorexigenic proopiomelanocortin (POMC) neurons in the hypothalamus to inhibit food intake. Because both NPY and POMC have also been shown to impact insulin action, we wondered whether PYY3-36 could improve insulin sensitivity. To address this question, we examined the acute effect of intravenous PYY3-36 on glucose and free fatty acid (FFA) flux during a hyperinsulinemic-euglycemic clamp in mice maintained on a high-fat diet for 2 weeks before the experiment. We also evaluated the effects of PYY3-36 infusion on glucose uptake in muscle and adipose tissue in this experimental context. Under basal conditions, none of the metabolic parameters were affected by PYY3-36. Under hyperinsulinemic conditions, glucose disposal was significantly increased in PYY 3-36-infused compared with vehicle-infused mice (103.8 ± 10.9 vs. 76.1 ± 11.4 μmol · min-1 · kg -1, respectively; P = 0.001). Accordingly, glucose uptake in muscle and adipose tissue was greater in PYY3-36-treated animals, although the difference with controls did not reach statistical significance in adipose tissue (muscle: 2.1 ± 0.5 vs. 1.5 ± 0.5 μmol/g tissue, P = 0.049; adipose tissue: 0.8 ± 0.4 vs. 0.4 ± 0.3 μmol/g tissue, P = 0.08). In contrast, PYY3-36 did not impact insulin action on endogenous glucose production or FFA metabolism. These data indicate that PYY3-36 reinforces insulin action on glucose disposal in mice fed a high-fat diet, through a mechanism that is independent of food intake and body weight. In contrast, it leaves glucose production and lipid flux largely unaffected in this experimental context.