Erythropoietin (EPO) has protective effects on many neurological diseases, including cerebral ischemia. Here, we aimed to test EPO's effects on ischemic neurovascular unit (NVU) injuries and examine whether the effects were dependent on connexin43 (Cx43) mediated gap junctional intercellular communication (GJIC). We detected the expression of Cx43 and phosphorylation of Cx43 (p-Cx43) at 1 d, 3 d, and 7 d after middle cerebral artery occlusion (MCAO). Meanwhile, we examined the effects of EPO on NVU injuries including neuronal survival, astrocyte activation and regeneration of endothelial cells as well as whether the effects were Cx43 dependent by using multiple inhibitors. We found EPO highly increased p-Cx43, but not total Cx43 at all chosen times. Importantly, EPO led to neurological and blood brain barrier functions improvement by associating with promotion of angiogenesis as well as reduction of neuronal death, astrocyte activation and neurotoxic substances levels. Moreover, these effects were significantly weakened by the inhibitors blocking GJIC, Cx43 communicative function, phosphorylation and expression, only Cx43 redistribution inhibitor excluded. Our data suggest the protective effects of EPO on NUV injuries are highly associated with the increase of p-Cx43, which improves GJIC to reduce neurotoxic substances. (C) 2015 Elsevier Inc. All rights reserved.