Studies with genetically modified insulinoma cells suggest that Group VIA Phospholipase A₂ (iPLA₂) participates in amplifying glucose-induced insulin secretion. INS-1 insulinoma cells that overexpress iPLA₂, for example, exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents. To determine whether similar effects occur in whole animals, we prepared transgenic mice in which the Rat Insulin 1 Promoter (RIP) drives iPLA₂ overexpression, and two characterized transgenic mouse lines exhibit similar phenotypes. Their pancreatic islet iPLA₂ expression is increased several-fold, as reflected by quantitative PCR of iPLA₂ mRNA, immunoblotting of iPLA₂ protein, and iPLA₂ enzymatic activity. Immunofluorescence microscopic studies of pancreatic sections confirm iPLA₂ overexpression in RIP-iPLA₂-transgenic (TG) islet -cells without obviously perturbed islet morphology. Male RIP-iPLA₂-TG mice exhibit lower blood glucose and higher plasma insulin concentrations than wild-type (WT) mice when fasting and develop lower blood glucose levels in glucose tolerance tests, but WT and TG blood glucose levels do not differ in insulin tolerance tests. Islets from male RIP-iPLA₂-TG mice exhibit greater amplification of glucose-induced insulin secretion by a cAMP-elevating agent than WT islets. In contrast, islets from male iPLA₂-null mice exhibit blunted insulin secretion, and those mice have impaired glucose tolerance. Arachidonate incorporation into and the phospholipid composition of RIP-iPLA₂-TG islets are normal, but they exhibit reduced Kv2.1 delayed rectifier current and prolonged glucose-induced action potentials and elevations of cytosolic [Ca²⁺] that suggest a molecular mechanism for the physiological role of iPLA₂ to amplify insulin secretion.