Glucagon-like peptide 2 (GLP-2) is an important regulator of intestinal growth and function. In adherable mixed meals the macronutrient composition with the best potential for stimulating GLP-2 secretion is not known. We compared the effect of 3 iso-energetic meals, where approximately 60 % of the energy ratio was provided as either carbohydrate, fat, or protein, respectively, on the post-prandial endogenous GLP-2 secretion. The responses were compared to secretion profiles of peptide YY (PYY), and glucose-dependent insulinotropic peptide (GIP). Ten healthy subjects were admitted on three occasions, at least a week apart, after a night of fasting. In an open-label, crossover design, they were randomized to receive a high carbohydrate (HC), high fat (HF) or high protein (HP) meal. The meals were approximately ∼3.9 MJ. Venous blood was collected for 240 min, and plasma concentrations of GLP-2, GIP and PYY were measured with specific radioimmunoassays. Mean GLP-2 levels peaked already at 30 min for the HC meal, however the HP meal induced the highest mean GLP-2 peaking levels, resulting in significantly higher mean GLP-2 area under the curve (AUC) from baseline of 7279 pmol*min/L, 95 %-CI [6081;8477] compared to the HC meal: 4764 pmol*min/L, 95 %-CI [3498;6029], p = 0.020 and the HF meal: 4796 pmol*min/L, [3385;6207], p = 0.011. Findings were similar for the PYY. The HC meal provided a greater AUC for GIP compared to the HP- and HF meals. The HP meal was most effective with respect to stimulation of the postprandial GLP-2 and PYY secretion, whereas the HC meal was more effective for GIP.