The omega-3 fatty acid, alpha linolenic acid (ALA) found in plant-derived foods induces significant cardiovascular benefits when ingested. ALA may be cardioprotective during ischemia; however, the mechanism(s) responsible for this effect is unknown. Isolated adult rat cardiomyocytes were exposed to medium containing ALA for 24 h and then exposed to non-ischemic (control), simulated ischemia (ISCH), or simulated ischemia/reperfusion (IR) conditions. Cardiomyocyte phospholipids were extracted and analyzed by an HPLC/electrospray ionization tandem mass spectrometry system. Pre-treatment of cells with ALA resulted in a significant incorporation of ALA within cardiomyocyte phosphatidylcholine. Cell death, DNA fragmentation and caspase-3 activity increased during ischemia and ischemia/reperfusion. Two pro-apoptotic oxidized phosphatidylcholine (OxPC) species, 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) were significantly increased during both ischemia and ischemia/reperfusion. Pre-treatment of the cells with ALA resulted in a significant reduction in cell death during ischemia and ischemia/reperfusion challenge. Apoptosis was also inhibited during ischemia and ischemia/reperfusion as shown by reduced DNA fragmentation and decreased caspase activation. ALA pre-treatment significantly decreased the production of POVPC and PGPC during ischemia and ischemia/reperfusion. ALA pre-treatment also significantly increased in resting Ca2+ during ischemia or ischemia/reperfusion but did not improve Ca2+ transients. ALA protects the cardiomyocyte from apoptotic cell death during simulated ISCH and IR by inhibiting the production of specific pro-apoptotic OxPC species. OxPCs represent a viable interventional target to protect the heart during ischemic challenge.