Aims/hypothesis Gastric inhibitory polypeptide (GIP) receptor antagonism with (Pro(3))GIP improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure/function in ob/ob mice. This study examined the ability of (Pro(3))GIP to counter the development of obesity, insulin resistance and diabetes in mice fed high-fat and cafeteria diets. Materials and methods Young Swiss TO mice on standard chow or high-fat, cafeteria or high-carbohydrate diets received daily injections of either saline or (Pro(3))GIP (25 nmol kg(-1)day(-1)) over 16 weeks. Food intake, body weight, and circulating glucose and insulin were measured frequently. At 16 weeks, glucose tolerance, insulin sensitivity, HbA(1c), circulating hormones and plasma lipids were assessed. Adipose tissue, liver and muscle were excised and weighed, and their histology and triacylglycerol content were further examined. Results (Pro(3) )GIP significantly reduced body weight, enhanced locomotor activity, and improved HbA(1c), glucose tolerance, beta cell responsiveness and insulin sensitivity in mice fed high-fat and cafeteria diets (p<0.05 to p<0.01). Similarly, (Pro 3)GIP significantly reduced plasma corticosterone and triacylglycerols (p<0.05 to p<0.001), while glucagon, resistin and adiponectin were unchanged. (Pro(3)) GIP decreased adipose tissue mass (p<0.01) and the triacylglycerol content of liver, muscle and adipose tissue (p<0.01 to p<0.001). Adipocyte size and liver morphology were partially normalised. (Pro(3))GIP did not significantly affect any of these parameters in mice fed a high-carbohydrate diet. Conclusions/interpretation (Pro(3))GIP protects against obesity, insulin resistance, glucose intolerance and associated disturbances in mice fed high-fat and cafeteria diets. This highlights chemical GIP receptor antagonism as a new possibility for the treatment of obesity and associated metabolic disturbances.