Background and purpose: Antagonism of the gastric inhibitory polypeptide (GIP) receptor with daily injection of proline-3 gastric inhibitory polypeptide ((Pro 3) GIP) can reverse or prevent many of the metabolic abnormalities associated with diet-induced obesity-diabetes (diabesity). This study has examined the ability of a novel and longer-acting form of (Pro 3) GIP, (Pro 3) GIP mini-polyethylene glycol ((Pro 3) GIP[ mPEG]), to counter diet-induced diabesity in mice, using a daily and intermittent dosing regime. Experimental approach: We studied the actions of (Pro 3) GIP[ mPEG] at the GIP receptor in vitro and in vivo in both dietary and genetic diabesity. Key results: (Pro 3) GIP[ mPEG] was completely resistant to degradation by dipeptidyl peptidase IV. (Pro 3) GIP[mPEG] inhibited GIP-induced cAMP and insulin production in vitro. A greater and prolonged antagonism of GIP-induced glucose-lowering action was followed (Pro 3) GIP[mPEG] administration, compared with (Pro 3) GIP. In contrast with (Pro 3) GIP, mice injected once every 3 days for 48 days with (Pro 3) GIP[mPEG] displayed reduced body weight gain and hyperinsulinemia with improved glucose tolerance and insulin secretory responses, compared with high-fat-fed controls. Daily i.p. injection of (Pro 3) GIP, (Pro 3) GIP[mPEG] or (Pro 3) GIP b.i.d. for 21 days also decreased body weight, circulating plasma insulin levels and improved glucose tolerance, compared with high-fat controls. Plasma triglycerides were decreased by (Pro 3) GIP[mPEG] and (Pro 3) GIP b.i.d. treatment groups. The observed changes were accompanied by enhancement of insulin sensitivity in all treatment regimes. (Pro 3) GIP[mPEG] was also effective over 16 days treatment of genetically obese-diabetic ob/ob mice. Conclusions and implications: These data demonstrate the utility of GIP receptor antagonism for the treatment of diabesity and the potential offered by (Pro 3) GIP[mPEG] as a long-acting stable GIP receptor antagonist.