To examine the role of group VIA phospholipase A2 (iPLA2 β) in specific cell lineages in insulin secretion and insulin action, we prepared mice with a selective iPLA2 β deficiency in cells of myelomonocytic lineage, including macrophages (MØ-iPLA2 β-KO), or in insulin-secreting β-cells (β-Cell-iPLA2 β-KO), respectively. MØ-iPLA2 β-KO mice exhibited normal glucose tolerance when fed standard chow and better glucose tolerance than floxed-iPLA2 β control mice after consuming a high-fat diet (HFD). MØ-iPLA2 β-KO mice exhibited normal glucose-stimulated insulin secretion (GSIS) in vivo and from isolated islets ex vivo compared to controls. Male MØ-iPLA2 β-KO mice exhibited enhanced insulin responsivity vs. controls after a prolonged HFD. In contrast, β-cell-iPLA2 β-KO mice exhibited impaired glucose tolerance when fed standard chow, and glucose tolerance deteriorated further when introduced to a HFD. β-Cell-iPLA2 β-KO mice exhibited impaired GSIS in vivo and from isolated islets ex vivo vs. controls. β-Cell-iPLA2 β-KO mice also exhibited an enhanced insulin responsivity compared to controls. These findings suggest that MØ iPLA2 β participates in HFD-induced deterioration in glucose tolerance and that this mainly reflects an effect on insulin responsivity rather than on insulin secretion. In contrast, β-cell iPLA2 β plays a role in GSIS and also appears to confer some protection against deterioration in β-cell functions induced by a HFD.