Ceramides are bioactive lipids that can promote splicing of apoptosis-related genes, including caspase 9 and BCL-x. A recent study demonstrated that expression of neutral sphingomyelinase (NSMase), an enzyme that hydrolyzes sphingomyelins to generate ceramide, is regulated by Group VIA phospholipase A2 (iPLA2β)-dependent mechanism during β-cell apoptosis. This prompted us to hypothesize that iPLA2 is upstream of ceramide generation in the process regulating splicing of apoptotic genes. To test this, Jurkat T cells were treated with the selective inhibitor of iPLA2β, bromoenol lactone (BEL), RNA was isolated and converted to cDNA, and caspase 9 and BCL-x mRNA viii species were amplified using RT-PCR. Inhibition of iPLA2β activity with BEL caused a significant shift in splicing favoring variants encoding the anti-apoptotic forms of caspase 9 (caspase 9b) and BCL-x (BCL-x(L)). This shift was consistent with previously reported effects of ceramide and suggested that iPLA2β regulates splicing of these pre-mRNAs. We next determined whether iPLA2β regulates splicing events during a biological response. Caspase-9 and BCL-x splice variants were compared in human and mouse islets, mouse islet cell lines, and in rat insulinoma (INS1) cells. INS-1 insulinoma cells were treated with thapsigargin to induce ER stress, which can eventually lead to apoptosis. Thapsigarin-treated INS-1 cells exhibited an increase in the ratio of BCL-x(s) (pro-apoptotic) to BCL-x(L) (anti-apoptotic) but BEL prevented this shift in splicing. Splicing data obtained from genetically modified rodent mice (iPLA2β knockouts and transgenics) also demonstrated the involvement of iPLA2β in alternative splicing. Together, these observations indicate that iPLA2β plays an important role in the regulation of pre-mRNA splicing of key apoptotic factors. Our findings therefore suggest a novel role for iPLA2β in determining whether cells survive or undergo apoptosis.