Glucagon is the physiological antagonist of insulin. Postprandial (pp) hyperglycemia in impaired glucose tolerance (IGT) and in type 2 diabetes mellitus (T2DM) may also depend on irregularities in glucagon secretion. This study investigated the glucagon excursion after a lipid-glucose-protein tolerance test in subjects with different stages of glucose intolerance. We also analyzed the relationship between pp glucagon secretion and hyperglycemias. A total of 64 men (27 healthy subjects with normal glucose tolerance [NGT], 15 with IGT, and 22 with T2DM) were examined. Plasma glucose (PG), insulin, proinsulin, free fatty acids, and triglycerides were measured in the fasting state and at 30 minutes and 2, 3, 4, and 6 hours after the intake of the test meal, which contained 126 g carbohydrates, 92 g fat, and 17 g protein. Postprandial concentrations of metabolic parameters were calculated as area under the curve (AUC). Glucagon was measured in the fasting state and at 30 minutes and 2 and 4 hours pp. Early glucagon increment was defined as glucagon at 30 minutes minus fasting glucagon. The insulin response was quantified as insulin increment divided by PG increment in the corresponding time. Insulin resistance was calculated using lomeostasis model assessment (HOMA). Fasting glucagon was significantly increased in IGT vs NGT (P < .05), and early glucagon increment was significantly higher in T2DM vs NGT and IGT (P < .05). The 2-hour glucagon concentration after the load (AUC) was increased in IGT and T2DM vs NGT (P < .05). Early glucagon increment and the 2-hour AUC of glucagon were strongly correlated to pp glycemia (r = 0.494 and P = .001, and r = 0.439 and P = .003, respectively). An inverse correlation was observed between early glucagon increment and insulin response at 30 minutes and 2 hours after the meal load (r = −0.287 and P = .026, and r = −0.435 and P = .001, respectively). The 2-hour AUC of glucagon was significantly associated with insulin resistance (r = 0.354, P = .020). Multivariate analysis revealed 2-hour insulin response and early glucagon increment as significant independent determinants of the AUC of PG in IGT (R = 0.787). In T2DM, 2-hour insulin response, insulin resistance, and early glucagon increment were significant determinants of the AUC of PG (R = 0.867). Our study suggests an important role for the irregularities in glucagon response in the pp glucose excursion after a standardized oral mixed meal in IGT and in T2DM. According to our data, a bihormonal imbalance starts before diabetes is diagnosed. Prospective studies are needed to evaluate the impact of glucagon on the progression of glucose intolerance and the possible effects of medicinal suppression of glucagon increment to prevent the progression of glucose tolerance.