We previously found that chronic exposure to glibenclamide inhibits acute glibenclamide-induced insulin secretion by reducing the number of functional ATP-sensitive K⁺ (K_ATP) channels on the plasma membrane of pancreatic β-cells. In the present study, we compared sulfonylurea-induced and glinide-induced insulin secretion in pancreatic β-cells chronically exposed to these widely used oral hypoglycemic agents. Chronic exposure of pancreatic β-cells to sulfonylureas (glibenclamide or tolbutamide) and glinide (nateglinide) similarly impaired their acute effectiveness by reducing the insulin content and the number of functional K_ATP channels on the plasma membrane. Functional expression of the voltage-dependent Ca²⁺ channels (VDCCs), ion channels that play a critical role in the K_ATP channel dependent insulin secretory pathway, was similar to that in drug-untreated cells. Chronic exposure to each of the three agents similarly accelerated apoptotic β-cell death. Thus, reduction of the insulin content, reduction of the number of functional K_ATP channels on the plasma membrane, and acceleration of apoptotic β-cell death all are involved in impaired insulinotropic agent-induced acute insulin secretion in the chronic phase of sulfonylurea and glinide treatment. These findings help to clarify the mechanism of secondary failure after long-term therapy by these hypoglycemic agents, and should have important clinical implications regarding pharmacotherapy for type 2 diabetes.