Ischemic stroke is an acute cerebrovascular disease. Currently, it is treated mainly by timely thrombolysis or mechanical thrombectomy to achieve vascular recanalization, but this process causes serious reperfusion injury to cerebrovasculature, damages the structure and function of the blood-brain barrier (BBB), and increases the risk of cerebral hemorrhage. Studies have shown that the Wnt/β-catenin signaling pathway plays a critical role in regulating the function of BBB, but whether the R-spondin, an agonistic protein of the Wnt/β-catenin signaling pathway, plays a role in regulation of BBB and cerebral injury is unclear. In this study, using mouse recombinant R-spondin-1 protein prepared in vitro, we determined the activation of Wnt/β-catenin signaling pathway induced by combined treatment of the R-spondin-1 protein and Wnt3a protein in mouse primary cerebrovascular endothelial cells, and found that R-spondin1 significantly changed the expression levels of BBB function related genes Cldn3 and Plvap. In a mouse cerebral ischemia/reperfusion model, intravenous injection of recombinant R-spondin-1 protein was inclined to reduce the cerebral infarction and increase mouse survival but did not reach statistical significance compared to phosphate buffer solution controls. Our study reported the molecular mechanisms of BBB function regulation by R-spondin protein, and preliminarily identified its therapeutic effect on cerebral injury following stroke and potential for clinical use.