Enzyme-resistant glucagon-like peptide-1 (GLP-1) receptor agonists and GIP receptor antagonists have been proposed to have therapeutic potential for the treatment of type 2 diabetes. Such benefits are based on actions mediated primarily through stimulation of insulin secretion or alleviation of insulin resistance respectively. This study examined the long-term actions of the stable GLP-1 receptor agonist (D-Ala(8))GLP-1 and the GIP receptor antagonist (Pro(3))GIP alone and in combination in high fat-fed mice. Mice on high-fat diet for 155 days were injected once daily with (D-Ala(8))GLP-1 or (Pro(3))GIP (25 nmol/kg body weight) for 24 days. In the following 24-day period, half of the (Pro(3))GIP-treated mice were administered an additional dose of (D-Ala(8))GLP-1 (25 nmol/kg body weight), while the remaining mice continued their original treatment regimes. Daily intraperitoneal injections of (D-Ala(8))GLP-1 or (Pro(3))GIP restored glycaemic control to normal levels and significantly (p < 0.05) improved glucose tolerance compared with high-fat controls by day 24. Food intake and body weights were not affected. On day 48, all treatment groups displayed significantly improved glucose tolerance (p < 0.05) and insulin sensitivity (p < 0.001) compared with high-fat controls on day 48. HDL cholesterol levels were significantly increased in mice treated with (D-Ala(8))GLP-1 alone (p < 0.05) or in combination with (Pro(3))GIP (p < 0.01) compared with normal chow-fed controls. These results illustrate efficacy of (Pro(3))GIP and (D-Ala(8))GLP-1 for treatment of glucose intolerance and insulin resistance caused by high-fat feeding. Combination therapy appeared to have little benefit over either treatment alone.