Exaggerated secretion of glucagon-like peptide 1 (GLP-1) is important for postprandial glucose tolerance after Roux-en-Y gastric bypass (RYGB), whereas the role of glucose-dependent insulinotropic polypeptide (GIP) remains to be resolved. We aimed to explore the relative importance of endogenously secreted GLP-1 and GIP on glucose tolerance and beta-cell function after RYGB. We used DPP-4 inhibition to enhance concentrations of intact GIP and GLP-1 and the GLP-1 receptor antagonist exendin 9-39 (Ex-9) for specific blockage of GLP-1 actions. Twelve glucose tolerant patients were studied after RYGB in a randomized, placebo-controlled, four-day, cross-over study with standard mixed meal tests and concurrent administration of: placebo, oral sitagliptin, Ex-9 infusion or combined Ex-9/sitagliptin. GLP-1 receptor antagonism increased glucose excursions, clearly attenuated beta-cell function and aggravated postprandial hyperglucagonaemia compared with placebo, whereas sitagliptin had no effect despite 2-3-fold increased concentrations of intact GLP-1 and GIP. Similarly, sitagliptin did not affect glucose tolerance or beta-cell function during GLP-1R blockage. This study confirms the importance of GLP-1 for glucose tolerance after RYGB via increased insulin and attenuated glucagon secretion in the postprandial state, whereas amplification of the GIP signal (or other DPP-4 sensitive glucose lowering mechanisms) did not appear to contribute to the improved glucose tolerance seen after RYGB.