The death of insulin-secreting β-cells that causes type I diabetes mellitus (DM) occurs in part by apoptosis, and apoptosis also contributes to progressive β-cell dysfunction in type II DM. Recent reports indicate that ER stress-induced apoptosis contributes to β-cell loss in diabetes. Agents that deplete ER calcium levels induce β-cell apoptosis by a process that is independent of increases in [Ca²⁺]_i. Here we report that the SERCA inhibitor thapsigargin induces apoptosis in INS-1 insulinoma cells and that this is inhibited by a bromoenol lactone (BEL) inhibitor of group VIA calcium-independent phospholipase A₂ (iPLA₂β). Overexpression of iPLA₂β amplifies thapsigargin-induced apoptosis of INS-1 cells, and this is also suppressed by BEL. The magnitude of thapsigargin-induced INS-1 cell apoptosis correlates with the level of iPLA₂β expression in various cell lines, and apoptosis is associated with stimulation of iPLA₂β activity, perinuclear accumulation of iPLA₂β protein and activity, and caspase-3-catalyzed cleavage of full-length 84 kDa iPLA₂β to a 62 kDa product that associates with nuclei. Thapsigargin also induces ceramide accumulation in INS-1 cells, and this response is amplified in cells that overexpress iPLA₂β. These findings indicate that iPLA₂β participates in ER stress-induced apoptosis, a pathway that promotes β-cell death in diabetes.