OBJECTIVE - We tested the hypotheses that in nondiabetic individuals, partial inhibition of insulin secretion with the ATP-sensitive K+ channel agonist (opener) diazoxide, compared with placebo, results in higher plasma glucose and higher plasma glucagon concentrations after a mixed meal and after administration of the sulfonylurea glimepiride. RESEARCH DESIGN AND METHODS - Plasma glucose, insulin, C-peptide, and glucagon concentrations were measured every 30 min from -60 through 180 min with random-sequence, double-blind administration of diazoxide (6.0 mg/kg) or placebo at -30 and 1 min, ingestion of a formula mixed meal (Ensure Plus) at 0 min after diazoxide and after placebo and, on a separate occasion, ingestion of glimepiride (4.0 mg) at 0 min (with glucose infused to prevent hypoglycemia) after diazoxide and after placebo in 11 healthy young adults. RESULTS - With diazoxide administration, insulin (P = 0.0016) and C-peptide (P = 0.0287) concentrations were decreased and glucose concentrations were increased (e.g., 180-min values of 106 ± 4 mg/dL [5.9 ± 0.2 mmol/L] compared with 87 ± 2 mg/dL [4.8 ± 0.1 mmol/L] with placebo; P < 0.0001), but glucagon concentrations were no different after the mixed meal. Similarly, with diazoxide, C-peptide concentrations were decreased (P = 0.0015) and glucose concentrations were increased (P < 0.0001), but glucagon concentrations declined similarly after glimepiride administration. CONCLUSIONS - Partial inhibition of insulin secretion results in impairment of glucose tolerance after a mixed meal and after glimepiride administration in the absence of a difference in glucagon secretion. They underscore the primary glucoregulatory role of insulin and support the evidence that β-cell secretion is not the only regulator of α-cell glucagon secretion.