Type 2 diabetes (T2D) is characterized by insulin resistance and pancreatic β-cell dysfunction, the latter possibly caused by a defect in insulin signaling in β-cells. We hypothesized that insulin's effect to potentiate glucose-stimulated insulin secretion (GSIS) would be diminished in insulin-resistant persons. To evaluate the effect of insulin to modulate GSIS in insulin-resistant compared with insulin-sensitive subjects, 10 participants with impaired glucose tolerance (IGT), 11 with T2D, and 8 healthy control subjects were studied on two occasions. The insulin secretory response was assessed by the administration of dextrose for 80 min following a 4-h clamp with either saline infusion (sham) or an isoglycemic-hyperinsulinemic clamp using B28-Asp-insulin (which can be distinguished immunologically from endogenous insulin) that raised insulin concentrations to high physiologic concentrations. Pre-exposure to insulin augmented GSIS in healthy persons. This effect was attenuated in insulin-resistant cohorts, both those with IGT and those with T2D. Insulin potentiates glucose-stimulated insulin secretion in insulin-resistant subjects to a lesser degree than in normal subjects. This is consistent with an effect of insulin to regulate β-cell function in humans in vivo with therapeutic implications.