Purpose. In this report, we investigated the protective mechanism of scutellarin (SCU) in vitro and in vivo which could be involved in endothelial cGMP-dependent protein kinase (PKG), vasodilator stimulated phosphoprotein (VASP) pathway, and vascular endothelium dysfunction (EtD). Method. Human brain microvascular endothelial cells (HBMECs) with hypoxia reoxygenation (HR) treatment and rats with cerebral ischemia reperfusion (CIR) treatment were applied. Protein and mRNA expression of PKG, VASP, and p-VASP were evaluated by Western blot and RT-PCR methods. Vascular EtD was assessed by using wire myography to determine endothelium-dependent vasorelaxation in isolated rat basilar artery (BA). Result. In cultured HBMECs, SCU (0.1, 1, and 10 mu M) increased cell viability, mRNA, protein level, and phosphorylative activity of PKG and VASP against HR injury. In HR model of BA, SCU increased protein level of P-VASP. In rat CIR model, wire myography demonstrated that SCU (45 and 90 mg/kg, i.v.) significantly reduced ischemic size by partially restoring the endothelium dependent vasodilation of BA; PKG inhibitor Rp-8-Br-cGMPS (50 mu g/kg, i.v.) reversed this protection of SCU in CIR rats. Conclusion. SCU protects against cerebral vascular EtD through endothelial PKG pathway activation.