Monounsaturated fatty acids, such as oleic acid (OA), and certain milk proteins, especially whey protein (WP) have insulinotropic effects and can reduce postprandial glycaemia. This effect may involve the incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). To explore this, we examined the release and inactivation of GIP and GLP-1, following administration of glucose with or without OA or WP through gastric gavage in anesthetized C57BL/6J mice. Insulin responses to glucose (75 mg) were 3-fold augmented by addition of WP (75 mg; P < 0.01), which was associated with enhanced oral glucose tolerance (P < 0.01). The insulin response to glucose was also augmented by addition of OA (34 mg; P < 0.05), although only 1.5-fold and with no associated increase in glucose elimination. The slope of the glucose-insulin curve was increased by OA (1.7-fold, P < 0.05) and by WP (4-fold, P < 0.01) compared with glucose alone, suggesting potentiation of glucose-stimulated insulin release. WP increased GLP-1 secretion (P < 0.01) while GIP secretion was unaffected. OA did not affect GIP or GLP-1 secretion. Nevertheless WP increased the levels of both intact GIP and intact GLP-1 (both P < 0.01) and OA increased the levels of intact GLP-1 (P < 0.05). WP inhibited dipeptidyl peptidase IV (DPP-4) activity in the proximal small intestine by 50% (P < 0.05), suggesting that luminal degradation of WP generates small fragments, which are substrates for DPP-4 and act as competitive inhibitors. We therefore conclude that fat and protein may serve as exogenous regulators of secretion and inactivation of the incretin hormones with beneficial influences on glucose metabolism.