High-mobility group box-1 (HMGB1) is a nuclear factor released by necrotic cells and by activated immune cells. HMGB1 signals via members of the toll-like receptor family and the receptor for advanced glycation end products (RAGE). Although HMGB1 has been implicated in ischemia/reperfusion (I/R) injury of the liver and lung, its role in I/R injury of the heart remains unclear. Here, we demonstrate that HMGB1 acts as an early mediator of inflammation and organ damage in I/R injury of the heart. HMGB1 levels were already elevated 30 minutes after hypoxia in vitro and in ischemic injury of the heart in vivo. Treatment of mice with recombinant HMGB1 worsened I/R injury, whereas treatment with HMGB1 box A significantly reduced infarct size and markers of tissue damage. In addition, HMGB1 inhibition with recombinant HMGB1 box A suggested an involvement of the mitogen-activated protein kinases jun N-terminal kinase and extracellular signal-regulated kinase 1/2, as well as the nuclear transcription factor nuclear factor-kappaB in I/R injury. Interestingly, infarct size and markers of tissue damage were not affected by administration of recombinant HMGB1 or HMGB1 antagonists in RAGE(-/-) mice, which demonstrated significantly reduced damage in reperfused hearts compared with wild-type mice. Coincubation studies using recombinant HMGB1 in vitro induced an inflammatory response in isolated macrophages from wild-type mice but not in macrophages from RAGE(-/-) mice. HMGB1 plays a major role in the early event of I/R injury by binding to RAGE, resulting in the activation of proinflammatory pathways and enhanced myocardial injury. Therefore, blockage of HMGB1 might represent a novel therapeutic strategy in I/R injury.