AAV-PHP.eB depends on endothelial cells to highly transduce the central nervous system (CNS) and is widely used for intravenous gene therapy. However, the transduction profile and therapeutic efficiency after endothelial cell injury such as ischemic stroke is largely unknown. In this study, we tested the transduction profiles of AAV-PHP.eB and developed intravenous NeuroD1 gene therapy to treat ischemic stroke in mice. We found that AAV-PHP.eB-GFP control virus crossed the BBB and infected brain cells efficiently in normal brain. However, after stroke, AAV-PHP.eB-GFP control virus was highly restricted in the blood vessels. Surprisingly, after switching to therapeutic vector AAV-PHP.eB-NeuroD1-GFP, the viral vector successfully crossed blood vessels and infected brain cells. Using Tie2-cre transgenic mice, we demonstrated that NeuroD1 regulated endothelial gene expression to modulate AAV-PHP.eB transduction. Following the changes of signaling pathways in endothelial cells, NeuroD1 effectively protected BBB integrity, attenuated neuroinflammation, inhibited neuron apoptosis and rescued motor deficits after ischemic stroke. Moreover, NeuroD1 over-expression in brain cells further promoted neural regeneration. These results indicate that intravenous gene therapy using AAV-PHP.eB for ischemic stroke differs from intracranial gene therapy and NeuroD1 intravenous delivery using AAV-PHP.eB efficiently rescue both vascular damage and neuronal loss, providing an advancing therapeutic treatment for stroke.