AIMS: Patients who have undergone Roux-en-Y gastric bypass surgery (GB) have exaggerated postprandial insulin secretion, which has been attributed to increased meal glucose appearance and enhanced incretin effect. Here, we sought to determine β-cell glucose sensitivity in the absence of meal stimulation and insulinotropic gut factors. MATERIALS AND METHODS: A total of 12 non-diabetic subjects with prior GB, and 7 matched non-surgical control subjects with normal glucose tolerance were studied. Blood glucose and insulin secretion rates were measured during a graded glucose infusion at increasing and then decreasing rates. Insulin sensitivity (SI ) and glucose effectiveness (SG ) were determined by the minimal model. RESULTS: GB subjects had SI comparable to that of control subjects. GB subjects had relative hyperglycaemia during the highest dose of glucose infusion associated with significantly reduced β-cell glucose sensitivity throughout both step-up (GB: 34 ± 6, CN: 82 ± 9 pmol min-1 mM-1 L, P < .0001) and step-down (GB: 31 ± 6, CN: 74 ± 9 pmol min-1 mM-1 L, P < .0001) phases of the glucose infusion. GB subjects also had reduced SG (GB: 0.04 ± 0.00, CN: 0.07 ± 0.01 min-1 , P = .004). CONCLUSION: In the absence of enteric stimuli, β-cell sensitivity to changes in glycaemia is blunted among individuals with GB, indicating a significant shift in a fundamental property of β-cell function several years after surgery.