The precise role of fat in postprandial glycemia and insulinemia has not been thoroughly researched because postprandial blood glucose and concurrent insulin secretion are largely assumed to be proportional to carbohydrate intake. Recent studies have suggested that dietary fat differentially regulates the postprandial insulin response. To explore this, we examined the effects of coadministered fat on glucose-induced glycemia and insulinemia in C57BL/6J mice. The insulin response to glucose was augmented by the addition of glycerol trioleate (TO) in a dose-dependent manner, which was associated with enhanced glucose transport from the circulation to muscle and adipose tissues. To investigate the mechanism underlying fat-induced hyperinsulinemia, we examined the release of the incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1. TO increased GIP secretion, whereas glucagon-like peptide-1 secretion was unaffected. TO-induced hyperinsulinemia was significantly attenuated by the pretreatment of mice with a specific GIP antagonist. Diacylglycerol (DAG) promoted lower postprandial GIP and triglyceride responses and, when ingested with glucose, a lower insulin response compared with triacylglycerol of a similar fatty acid composition. Pluronic L-81, an inhibitor of chylomicron formation, reduced not only the triglyceride response but also TO-induced GIP secretion, indicating that the lower GIP response after DAG ingestion may be associated with retarded chylomicron formation in the small intestine. We conclude that dietary fat augments glucose-induced insulinemia via gut-derived GIP and, thereby, influences postprandial nutrient metabolism in mice. DAG promotes a lower GIP and thereby reduced insulin responses compared with triacylglycerol, which may differentially influence postprandial energy homeostasis.