The involvement of the group VI iPLA₂s (iPLA₂-VI) in in vitro ischemia (OGD, oxygen and glucose deprivation) in mouse C2C12 myotubes have been investigated. OGD induced a time-dependent (0-6 h) increase in BEL-sensitive iPLA₂ activity which was suppressed by specific siRNA knockdown of iPLA₂-VIA. OGD was associated with an increase in iPLA₂-VIA protein level whereas the mRNA level was unchanged. The level of iPLA₂-VIB mRNA and protein were not increased by OGD. RT-PCR and Western blotting identified a mouse iPLA₂-VIA homolog to the catalytically inactive 50 kDa iPLA₂-VIA-Ankyrin variants previously identified in human. Both the mRNA and protein levels of this ~ 50 kDa variant were reduced significantly within 1 h following OGD. In C2C12 myoblasts, iPLA₂-VIA seemed to predominantly reside at the endoplasmatic reticulum (ER), where it accumulated further during OGD. A time-dependent reduction in cell viability during the early OGD period (3 h) was partially prevented by iPLA₂-VIA knockdown or pharmacological inhibition (BEL 10 µM), whereas iPLA₂-VIA overexpression had no effect on cell viability. Altogether, these data demonstrate that OGD in C2C12 myotubes is associated with an increase in iPLA₂-VIA activity which decrease cell viability. iPLA₂-VIA activation may be modulated by changes in the level of active and inactive iPLA₂-VIA isoforms.