The hematopoietic growth factor erythropoietin (EPO) attracts attention due to its all-tissue-protective pleiotropic properties. We studied the effect of EPO on dermal regeneration using intravital microscopy in a model of full dermal thickness wounds in the skin-fold chamber of hairless mice. Animals received repetitive low doses or high doses of EPO (RLD-EPO or RHD-EPO) or a single high dose of EPO (SHD-EPO). SHD-EPO accelerated wound epithelialization, reduced wound cellularity, and induced maturation of newly formed microvascular networks. In contrast, RHD-EPO impaired the healing process, as indicated by delayed epithelialization, high wound cellularity, and lack of maturation of microvascular networks. Also, RHD-EPO caused an excessive erythrocyte mass and rheological malfunction, further deteriorating vessel and tissue maturation. Moreover, RHD-EPO altered fibroblast and keratinocyte migration in vitro, while both cell types exposed to RLD-EPO, and, in particular, to SHD-EPO showed accelerated wound scratch closure. In summary, our data show that a single application of a high dose of EPO accelerates and improves skin wound healing.