This study examined the glucose-lowering and insulinotropic properties of acylated GLP-1 and GIP peptides in type 2 diabetes and obesity. GLP-1, GIP, Liraglutide, N-AcGIP(Lys37Myr), a simple combination of both peptides, and an overnight preparation of Liraglutide and N-AcGIP(Lys37Myr) (Lira-AcGIP preparation) were incubated with DPP-IV to assess peptide stability and BRIN-BD11 cells were used to evaluate cAMP production and insulin secretion. Acute glucose-lowering and insulinotropic actions were evaluated in Swiss TO mice. Sub-chronic studies on glucose homeostasis, insulin secretion, food intake and bodyweight were evaluated in ob/ob mice. Liraglutide, N-AcGIP(Lys37Myr), a simple combination of both peptides and Lira-AcGIP preparation demonstrated improved DPP-IV resistance (P < 0.001), while stimulating cAMP production and insulin secretion (1.4 to 2-fold; P < 0.001). Lira-AcGIP preparation was more potent at lowering plasma glucose (20 to 51% reduction; P < 0.05 - P < 0.001) and stimulating insulin secretion (1.5- to 1.8-fold; P < 0.05 - P < 0.001) compared to Liraglutide and N-AcGIP(Lys37Myr) or a simple peptide combination. Daily administration of Lira-AcGIP preparation to ob/ob mice lowered bodyweight (7 to 9%; P < 0.05), food intake (23%; P < 0.05) and plasma glucose (46% reduction; P < 0.001) while increasing plasma insulin (1.5- to 1.6-fold; P < 0.001). Lira-AcGIP preparation enhanced glucose tolerance, insulin response to glucose and insulin content (P < 0.05 - P < 0.001). These data demonstrate that combined preparation of acylated GLP-1 and GIP peptides, Liraglutide and N-AcGIP(Lys37Myr), markedly improved glucose-lowering and insulinotropic properties in diabetes-obesity compared with either incretin mimetic given individually.