PYY_3-36 is a gut-derived hormone acting on hypothalamic nuclei to inhibit food intake. We recently showed that PYY_3-36 acutely reinforces insulin action on glucose disposal in mice. We now aimed to evaluate effects of PYY_3-36 on energy metabolism and the impact of chronic PYY_3-36 treatment on insulin sensitivity. Mice received a single injection of PYY_3-36 or were injected once daily for 7 days and energy metabolism was subsequently measured in a metabolic cage. Furthermore, the effects of chronic PYY_3-36 administration (continuous and intermittent) on glucose turnover were determined during a hyperinsulinemic-euglycemic clamp. PYY_3-36 inhibited cumulative food intake during 30 minutes of refeeding after an overnight fast (0.29 ± 0.04 vs. 0.56 ± 0.12 g, P=0.036) in an acute setting, but not after 7 days of daily dosing. Body weight, total energy expenditure and physical activity were not affected by PYY_3-36. However, it significantly decreased the respiratory quotient. Both continuous and intermittent PYY_3-36 treatment significantly enhanced insulin-mediated whole body glucose disposal in comparison with vehicle treatment (81.2 ± 6.2 vs. 77.1 ± 5.2 vs. 63.4 ± 5.5 µmol/min/kg, respectively). In particular, PYY_3-36 treatment increased glucose uptake in adipose tissue, whereas its impact on glucose disposal in muscle did not attain statistical significance. PYY_3-36 treatment shifts the balance of fuel use in favor of fatty acids and enhances insulin sensitivity in mice, where it particularly promotes insulin-mediated glucose disposal. Notably, these metabolic effects of PYY_3-36 remain unabated after chronic administration, in contrast to its anorexic effects.