Elevated glucagon is associated with fasting hyperglycemia in type 2 diabetes. We assessed the effects of the glucagon receptor antagonist (2R)-N-[4-({4-(1-cyclohexen-1-yl)[(3,5-dichloroanilino)carbonyl]anilino}methyl)benzoyl]-2-hydroxy-b-alanine (NNC 25-0926) on hepatic glucose production (HPG) in vivo, using arteriovenous difference and tracer techniques in conscious dogs. The experiments consisted of equilibration (–140 to –40 min), control (40–0 min), and experimental [0–180 min, divided into P1 (0–60 min) and P2 (60–180 min)] periods. In P1, NNC 25-0926 was given intragastrically at 0 (veh), 10, 20, 40, or 100 mg/kg, and euglycemia was maintained. In P2, somatostatin, basal intraportal insulin, and 5-fold basal intraportal glucagon (2.5 ng/kg/min) were infused. Arterial plasma insulin levels remained basal throughout the study in all groups. Arterial plasma glucagon levels remained basal during the control period and P1 and then increased to ∼70 pg/ml in P2 in all groups. Arterial plasma glucose levels were basal in the control period and P1 in all groups. In P2, the arterial glucose level increased to 245 ± 22 and 172 ± 15 mg/dl in the veh and 10 mg/kg groups, respectively, whereas in the 20, 40, and 100 mg/kg groups, there was no rise in glucose. Net hepatic glucose output was ∼2 mg/kg/min in all groups during the control period. In P2, it increased by 9.4 ± 2 mg/kg/min in the veh group. In the 10, 20, 40, and 100 mg/kg groups, the rise was only 4.1 ± 0.9, 1.6 ± 0.6, 2.4 ± 0.7, and 1.5 ± 0.3 mg/kg/min, respectively, due to inhibition of glycogenolysis. In conclusion, NNC 25-0926 effectively blocked the ability of glucagon to increase HGP in the dog.