Calcium-independent phospholipase A2 γ (iPLA2 γ ) is a mitochondrial enzyme that produces lipid second messengers that facilitate opening of the mitochondrial permeability transition pore (mPTP) and contribute to the production of oxidized fatty acids in myocardium. To specifically identify the roles of iPLA2 γ in cardiac myocytes, we generated cardiac myocyte-specific iPLA2 γ knock-out (CMiPLA2 γ KO) mice by removing the exon encoding the active site serine (Ser-477). Hearts of CMiPLA2 γ KO mice exhibited normal hemodynamic function, glycerophospholipid molecular species composition, and normal rates of mitochondrial respiration and ATP production. In contrast, CMiPLA2 γ KO mice demonstrated attenuated Ca 2+ -induced mPTP opening that could be rapidly restored by the addition of palmitate and substantially reduced production of oxidized polyunsaturated fatty acids (PUFAs). Furthermore, myocardial ischemia/reperfusion (I/R) in CMiPLA2 γ KO mice (30 min of ischemia followed by 30 min of reperfusion in vivo) dramatically decreased oxidized fatty acid production in the ischemic border zones. Moreover, CMiPLA2 γ KO mice subjected to 30 min of ischemia followed by 24 h of reperfusion in vivo developed substantially less cardiac necrosis in the area-atrisk in comparison with their WT littermates. Furthermore, we found that membrane depolarization in murine heart mitochondria was sensitized to Ca 2+ by the presence of oxidized PUFAs. Because mitochondrial membrane depolarization and calcium are known to activate iPLA2 γ , these results are consistent with salvage of myocardium after I/R by iPLA2 γ loss of function through decreasing mPTP opening, diminishing production of proinflammatory oxidized fatty acids, and attenuating the deleterious effects of abrupt increases in calcium ion on membrane potential during reperfusion.