Abstract During ischemia/reperfusion injury (I/RI), leukocytes release cytokines and exacerbate inflammation causing cardiac dysfunction, while neutrophils produce oxygen radicals that worsen the initial myocardial infarction. Phospholipase D is a cell membrane remodeling and signaling protein implicated in the pathology of I/RI. We analyzed I/RI by ejection fraction, infarct size and serum Troponin levels in wild-type, PLD1−/− and PLD2−/− mice. PLD-deficient mice or inhibition of PLD in WT mice protected against I/RI. Removing PLD2 yielded a slightly better outcome, suggesting PLD2 contributed more to the injury than PLD1, particularly at longer (>4 h) reperfusion times. Three cell signaling mechanisms were investigated: mTOR pathway, Aurora Kinase A and Cyclin-D3. Gene expression of the mTOR pathway was increased in response to I/RI in WT mice and abrogated in PLD-KO mice suggesting PLD augmented mTOR pathway during IR/I. PLD1−/− mice have lower expression of CCND3/Aurora Kinase A, while PLD2−/− mice expressed more phospho-CyclinD3. We detected neutrophil-associated Ly6G after adoptive transfer of bone marrow neutrophils during I/RI. Myocardial PMN accumulation was increased following I/R and PLD inhibitors abrogated PMN accumulation. Less myocardial PMN accumulation was observed in PLD-KO mice following IR/I. Adoptive transfer of fluorescently labeled PLD2−/− or PLD1−/−PMN into PLD1−/− or PLD2−/− mice, respectively, demonstrated that PLD1 containing (PLD2−/−) PMNs accumulated more significantly in the myocardium following I/RI. This study demonstrates for the first time the specific mechanistic contribution of mammalian PLD1 and PLD2 to MI/R injury in a murine model and their role in neutrophil infiltration.