Background— All treatments of acute myocardial infarction are aimed at rapid revascularization of the occluded vessel; however, no clinical strategies are currently available to protect the heart from ischemia/reperfusion injury after restitution of blood flow. We hypothesized that some of the cholesterol transport–independent biological properties of high-density lipoprotein (HDL) implied in atheroprotection may also be beneficial in settings of acute myocardial reperfusion injury. Methods and Results— In an in vivo mouse model of myocardial ischemia/reperfusion, we observed that HDL and its sphingolipid component, sphingosine-1-phosphate (S1P), dramatically attenuated infarction size by ≈20% and 40%, respectively. The underlying mechanism was an inhibition of inflammatory neutrophil recruitment and cardiomyocyte apoptosis in the infarcted area. In vitro, HDL and S1P potently suppressed leukocyte adhesion to activated endothelium under flow and protected rat neonatal cardiomyocytes against apoptosis. In vivo, HDL- and S1P-mediated cardioprotection was dependent on nitric oxide (NO) and the S1P ₃ lysophospholipid receptor, because it was abolished by pharmacological NO synthase inhibition and was completely absent in S1P ₃ -deficient mice. Conclusions— Our data demonstrate that HDL and its constituent, S1P, acutely protect the heart against ischemia/reperfusion injury in vivo via an S1P ₃ -mediated and NO-dependent pathway. A rapid therapeutic elevation of S1P-containing HDL plasma levels may be beneficial in patients at high risk of acute myocardial ischemia.