Studies involving pharmacologic or molecular biologic manipulation of Group VIA phospholipase A2 (iPLA2β) activity in pancreatic islets and insulinoma cells suggest that iPLA2β participates in insulin secretion. It has also been suggested that iPLA2β is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels and arachidonate incorporation into phosphatidylcholine (PC). We have generated iPLA2β-null mice by homologous recombination and have reported that they exhibit reduced male fertility and defective motility of spermatozoa. Here we report that pancreatic islets from iPLA2β-null mice have impaired insulin secretory responses to D-glucose and forskolin. Electrospray ionization mass spectrometric analyses indicate that the abundance of arachidonate-containing PC species of islets, brain, and other tissues from iPLA2β-null mice is virtually identical to that of wild-type mice, and no iPLA2β mRNA was observed in any tissue from iPLA2β-null mice at any age. Despite the insulin secretory abnormalities of isolated islets, fasting and fed blood glucose concentrations of iPLA2β-null and wild-type mice are essentially identical under normal circumstances, but iPLA2β-null mice develop more severe hyperglycemia than wild-type mice after administration of multiple low doses of the β-cell toxin streptozotocin, suggesting an impaired islet secretory reserve. A high fat diet also induces more severe glucose intolerance in iPLA2β-null mice than in wild-type mice, but PLA 2β-null mice have greater responsiveness to exogenous insulin than do wild-type mice fed a high fat diet. These and previous findings thus indicate that iPLA2β-null mice exhibit phenotypic abnormalities in pancreatic islets in addition to testes and macrophages. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.