Recent findings have attested the protective effects of erythropoietin (EPO) in ischemically challenged organs. We therefore aimed at elaborating the underlying mechanism of EPO-mediated protection in musculocutaneous tissue undergoing persistent ischemia after acute injury. Mice were assigned to five experimental groups equipped with a randomly perfused flap fixed in a dorsal skinfold chamber, whereas the sixth group did not undergo flap preparation: EPO, L-Name, EPO and L-Name, EPO and bevacizumab, untreated flap, and nonischemic chamber (control). Intravital fluorescence microscopic analysis of microhemodynamics, apoptotic cell death, macromolecular leakage and angiogenesis was carried out over a 10-day period. Further, immunohistochemical analysis was used to study the protein expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF). Increased expression of eNOS in EPO-administered mice correlated with significant arteriolar dilation and thus increased blood flow resulting in a maintained functional capillary density (FCD) at day 10. In addition, EPO induced a VEGF upregulation, which was associated with newly formed capillaries. In addition, EPO was able to reduce ischemia-induced apoptotic cell death and finally to significantly reduce flap necrosis. In contrast, coadministration of L-Name abolished EPO-mediated tissue protection by abrogating the dilatory effect resulting in reduced FCD and tissue survival, without counteracting angiogenesis and apoptotic cell death, whereas additional administration of bevacizumab did not influence the beneficial effect of EPO on flap survival despite abrogating angiogenesis. Macromolecular leakage was found to be increased in all treatment groups. This study shows that EPO administration prevents musculocutaneous tissue from ischemic necrosis as a consequence of an eNOS-dependent arteriolar hyperperfusion maintaining capillary perfusion, thus representing a promising approach to pharmacologically protect ischemically challenged tissue.