We evaluated the acute effects of oxyntomodulin (OXM), a proglucagon derived peptide, on glucose metabolism in diet-induced insulin resistant male C57Bl/6 mice. To determine the effects on glucose tolerance, mice were intraperitoneally injected with OXM (0.75, 2.5, or 7.5 nmol) or vehicle prior to an intraperitoneal glucose tolerance test. 0.75 nmol/h OXM or vehicle was infused during a hyperinsulinemic euglycemic clamp to quantify insulin action on glucose production and disposal. OXM dose-dependently improved glucose tolerance as estimated by the area under the curve (AUC) for glucose (OXM, 7.5 nmol: 1564 ± 460, p<0.01; 2.5 nmol: 1828 ± 684, p<0.01; 0.75 nmol: 2322 ± 303, p<0.05; control: 2790 ± 222 mmol/l/120 min). Insulin levels in response to glucose administration were higher in 7.5 nmol OXM-treated animals compared to controls (AUC_insulin: 343 ± 113 vs. 231 ± 39 ng/ml/120 min, p<0.05). In basal clamp conditions, OXM increased endogenous glucose production (82.2 ± 14.7 vs. 39.9 ± 5.7 µmol/min/kg, p<0.001). During insulin infusion, insulin levels were twice as high in OXM-treated mice compared to controls (10.6 ± 2.8 vs. 4.4 ± 2.2 ng/ml, p<0.01). Consequently, glucose infusion rate (118.6 ± 30.8 vs. 38.8 ± 26.4 µl/h, p<0.001) and glucose disposal (88.1 ± 13.0 vs. 45.2 ± 6.9 µmol/min/kg, p<0.001) were enhanced in mice that received OXM. In addition, glucose production was more suppressed during OXM infusion (35.7 ± 15.5 vs. 15.8 ± 11.4% inhibition, p<0.05). However, if these data were expressed per unit concentration of circulating insulin, OXM did not affect insulin action on glucose disposal and production. These results indicate that OXM beneficially affects glucose metabolism in diet-induced insulin resistant C57Bl/6 mice. It ameliorates glucose intolerance, most likely because it elevates glucose-induced plasma insulin concentrations. OXM does not appear to impact on insulin action.