Postprandial secretion of insulin and glucose-dependent insulinotropic polypeptide (GIP) is differentially regulated by not only dietary carbohydrate but also fat. Recent studies have shown that the ingestion of diacylglycerol (DAG) results in lower postprandial insulin and GIP release than that of triacylglycerol (TAG), suggesting a possible mechanism for the antiobesity effect of DAG. The structural and metabolic characteristics of DAG are believed to be responsible for its beneficial effects. This study was designed to clarify the effect of 1-monoacylglycerol [oleic acid-rich (1-MO)], the characteristic metabolite of DAG, on postprandial insulin and GIP secretion, and the underlying mechanism. Dietary 1-MO dose dependently stimulated whole body fat utilization, and reduced high-fat diet-induced body weight gain and visceral fat accumulation in mice, both of which are consistent with the physiological effect of dietary DAG. Although glucose-stimulated insulin and GIP release was augmented by the addition of fat, coingestion of 1-MO reduced the postprandial hormone release in a dose-dependent manner. Either glucose or fatty acid transport into the everted intestinal sacs and enteroendocrine HuTu-80 cells was also reduced by the addition of 1-MO. Reduction of either glucose or fatty acid transport or the nutrient-stimulated GIP release by 1-MO was nullified when the intestine was pretreated with sodium-glucose cotransporter-1 (SGLT-1) or fatty acid translocase (FAT)/CD36 inhibitor. We conclude that dietary 1-MO attenuates postprandial GIP and insulin secretion by reducing the intestinal transport of the GIP secretagogues, which may be mediated via SGLT-1 and FAT/CD36. Reduced secretion of these anabolic hormones by 1-MO may be related to the antiobesity effect of DAG.