The role of phosphatidylinositol (PI) 3-kinase in the regulation of pancreatic β-cell function was investigated. PI 3-kinase activity in p85α regulatory subunit–deficient (p85α^−/−) islets was decreased to ∼20% of that in wild-type controls. Insulin content and mass of rough endoplasmic reticula were decreased in β-cells from p85α^−/− mice with increased insulin sensitivity. However, p85α^−/− β-cells exhibited a marked increase in the insulin secretory response to higher concentrations of glucose. When PI 3-kinase in wild-type islets was suppressed by wortmannin or LY294002, the secretion was also substantially potentiated. Wortmannin’s potentiating effect was not due to augmentation in glucose metabolism or cytosolic [Ca²⁺] elevation. Results of p85α^−/− islets and wortmannin-treated wild-type islets stimulated with diazoxide and KCl showed that inhibition of PI 3-kinase activity exerted its effect on secretion, at least in part, distal to a cytosolic [Ca²⁺] elevation. These results suggest that PI 3-kinase activity normally plays a crucial role in the suppression of glucose-stimulated insulin secretion.