Diabetes is a consequence of reduced β-cell function and mass, due to β-cell apoptosis. Endoplasmic reticulum (ER) stress is induced during β-cell apoptosis due to various stimuli, and our work indicates that group VIA phospholipase A2β (iPLA2β) participates in this process. Delineation of underlying mechanism(s) reveals that ER stress reduces the anti-apoptotic Bcl-x(L) protein in INS-1 cells. The Bcl-x pre-mRNA undergoes alternative pre-mRNA splicing to generate Bcl-x(L) or Bcl-x(S) mature mRNA. We show that both thapsigargin-induced and spontaneous ER stress are associated with reductions in the ratio of Bcl-x(L)/Bcl-x(S) mRNA in INS-1 and islet β-cells. However, chemical inactivation or knockdown of iPLA2β augments the Bcl-x(L)/Bcl-x(S) ratio. Furthermore, the ratio is lower in islets from islet-specific RIP-iPLA2β transgenic mice, whereas islets from global iPLA2β(-/-) mice exhibit the opposite phenotype. In view of our earlier reports that iPLA2β induces ceramide accumulation through neutral sphingomyelinase 2 and that ceramides shift the Bcl-x 5'-splice site (5'SS) selection in favor of Bcl-x(S), we investigated the potential link between Bcl-x splicing and the iPLA2β/ceramide axis. Exogenous C6-ceramide did not alter Bcl-x 5'SS selection in INS-1 cells, and neutral sphingomyelinase 2 inactivation only partially prevented the ER stress-induced shift in Bcl-x splicing. In contrast, 5(S)-hydroxytetraenoic acid augmented the ratio of Bcl-x(L)/Bcl-x(S) by 15.5-fold. Taken together, these data indicate that β-cell apoptosis is, in part, attributable to the modulation of 5'SS selection in the Bcl-x pre-mRNA by bioactive lipids modulated by iPLA2β.