Glucagon-like-peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon bind to related members of the same receptor superfamily and exert important effects on glucose homeostasis, insulin secretion and energy regulation. The present study assessed the biological actions and therapeutic utility of novel GIP/glucagon/GLP-1 hybrid peptides. Nine novel peptides were synthesized and exhibited complete DPP-IV resistance and enhanced in vitro insulin secretion. The most promising peptide, [DA2]GLP-1/GcG, stimulated cAMP production in GIP-, GLP-1- and glucagon-R transfected cells. Acute administration of [DA2]GLP-1/GcG in combination with glucose significantly lowered plasma glucose and increased plasma insulin in normal and obese diabetic (ob/ob) mice. Furthermore, [DA2]GLP-1/GcG elicited a protracted glucose-lowering and insulinotropic effect in high-fat fed mice. Twice-daily administration of [DA2]GLP-1/GcG for 21 days decreased body weight, non-fasting plasma glucose and increased circulating plasma insulin concentrations in high-fat fed mice. Furthermore, [DA2]GLP-1/GcG significantly improved glucose tolerance and insulin sensitivity by day 21. Interestingly, locomotor activity was increased in [DA2]GLP-1/GcG mice, without appreciable changes in aspects of metabolic rate. Studies in knockout mice confirmed the biological action of [DA2]GLP-1/GcG via multiple targets including GIP-, GLP-1- and glucagon-Rs. The data suggests significant promise for novel triple-acting hybrid peptides as therapeutic options for obesity-diabetes.