The gut hormone gastric inhibitory polypeptide (GIP) plays a key role in glucose homeostasis and lipid metabolism. This study investigated the effects of administration of a stable and specific GIP-R antagonist, (Pro³)GIP in mice previously fed a high fat diet for 160 days to induce obesity and related diabetes. Daily i.p. injection of (Pro³)GIP over 50 days significantly decreased body weight compared with saline-treated controls with a modest increase in locomotor activity but no change of high fat diet intake. Plasma glucose, glycated haemoglobin and pancreatic insulin were restored to levels of chow-fed mice, and circulating triglyceride and cholesterol were significantly decreased. (Pro³)GIP treatment also significantly decreased circulating glucagon and corticosterone but concentrations of GLP-1, GIP, resistin and adiponectin were unchanged. Adipose tissue mass, adipocyte hypertrophy and deposition of triglyceride in liver and muscle were significantly decreased. These changes were accompanied by significant improvement of insulin sensitivity, meal tolerance and normalisation of glucose tolerance in (Pro³)GIP treated high fat fed mice. (Pro³)GIP concentrations peaked rapidly and remained elevated 24 h after injection. These data indicate that GIP-receptor antagonism action using (Pro³)GIP provides an effective means of countering obesity and related diabetes induced by consumption of high fat energy rich diet.