OBJECTIVE: Remote ischemic preconditioning (RIPC) is an intervention process where the application of multiple cycles of short ischemia/reperfusion (I/R) in a remote vascular bed provides protection against I/R injury. However, the identity of the specific RIPC factor and the mechanism by which RIPC alleviates I/R injury remains unclear. Here, we have investigated the identity and the mechanism by which the RIPC factor provides protection. APPROACH AND RESULTS: Using fluorescent in situ hybridization and immunofluorescence, we found that RIPC induces Nrg1β expression in the endothelial cells, which is secreted into the serum. Whereas, RIPC protected against myocardial apoptosis and infarction, treatment with neutralizing-Nrg1 antibodies abolished the protective effect of RIPC. Further, increased superoxide anion generated in RIPC is required for Nrg1 expression. Improved myocardial perfusion and nitric oxide production were achieved by RIPC as determined by contrast echocardiography and electron spin resonance. However, treatment with neutralizing-Nrg1β antibody abrogated these effects, suggesting Nrg1β is a RIPC factor. ErbB2 (Erb-B2 receptor tyrosine kinase 2) is not expressed in the adult murine cardiomyocytes, but expressed in the endothelial cells of heart which is degraded in I/R. RIPC-induced Nrg1β interacts with endothelial ErbB2 and thereby prevents its degradation. Mitochondrial Trx2 (thioredoxin) is degraded in I/R, but rescue of ErbB2 by Nrg1β prevents Trx-2 degradation that decreased myocardial apoptosis in I/R. CONCLUSIONS: Nrg1β is a RIPC factor that interacts with endothelial ErbB2 and prevents its degradation, which in turn prevents Trx2 degradation due to phosphorylation and inactivation of ATG5 (autophagy-related 5) by ErbB2. Nrg1β also restored loss of eNOS (endothelial nitric oxide synthase) function in I/R via its interaction with Src.