Ablation of glucagon receptor signaling represents a potential treatment option for type 2 diabetes (T2DM). Additionally, activation of glucose-dependent insulinotropic polypeptide (GIP) receptor signaling also holds therapeutic promise for T2DM. Therefore, this study examined both independent and combined metabolic actions of desHis 1 Pro 4 Glu 9 (Lys 12 PAL)-glucagon (glucagon receptor antagonist) and d-Ala 2 GIP (GIP receptor agonist) in diet-induced obese mice. Glucagon receptor binding has been linked to alpha-helical structure and desHis 1 Pro 4 Glu 9 (Lys 12 PAL)-glucagon displayed enhanced alpha-helical content compared with native glucagon. In clonal pancreatic BRIN-BD11 beta-cells, desHis 1 Pro 4 Glu 9 (Lys 12 PAL)-glucagon was devoid of any insulinotropic or cAMP-generating actions, and did not impede d-Ala 2 GIP-mediated (P < 0.01 to P < 0.001) effects on insulin and cAMP production. Twice-daily injection of desHis 1 Pro 4 Glu 9 (Lys 12 PAL)-glucagon or d-Ala 2 GIP alone, and in combination, in highfat-fed mice failed to affect body weight or energy intake. Circulating blood glucose levels were significantly (P < 0.05 to P < 0.01) decreased by all treatments regimens, with plasma and pancreatic insulin elevated (P < 0.05 to P < 0.001) in all mice receiving d-Ala 2 GIP. Interestingly, plasma glucagon concentrations were decreased (P < 0.05) by sustained glucagon inhibition (day 28), but increased (P < 0.05) by d-Ala 2 GIP therapy, with a combined treatment resulting in glucagon concentration similar to saline controls. All treatments improved (P < 0.01) intraperitoneal and oral glucose tolerance, and peripheral insulin sensitivity. d-Ala 2 GIP-treated mice showed increased glucoseinduced insulin secretion in response to intraperitoneal and oral glucose. Metabolic rate and ambulatory locomotor activity were increased (P < 0.05 to P < 0.001) in all desHis 1 Pro 4 Glu 9 (Lys 12 PAL)-glucagon-treated mice. These studies highlight the potential of glucagon receptor inhibition alone, and in combination with GIP receptor activation, for T2DM treatment.