Scutellarin (SCU), a flavonoid glycoside compound, has been successfully used in clinic for treatment of ischemic diseases in China. In this report, we checked the effects of SCU on endothelium dysfunction (ED) of coronary artery (CA) against myocardial ischemia reperfusion (MIR) injury in vivo. The involvement of PKG-Iα was further studied using cultured endothelial cells subjected to hypoxia reoxygenation (HR) injury in vitro. In rat MIR model, SCU (45 and 90 mg/kg, iv) significantly reduced ischemic size and restored the endothelium-dependent vasodilation of isolated CA rings. PKG inhibitor Rp-8-Br-cGMP (50 μg/kg, iv) could ameliorate the protective effects of SCU. Increase in phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a main substrate of PKG, at Ser 239 was observed in both heart tissue and serum of SCU-treated animals. In cultured human cardiac microvascular endothelial cells (HCMECs), SCU (1 and 10 μM) dose-dependently protected cell viability and increased the mRNA and protein level of PKG-Iα against HR injury. The activity of PKG was also increased by SCU treatment. The activation of PKG-1α was then studied using targeted proteomic analysis (MRM-MS) checking the phosphorylation state of the autophosphorylation domain (aa42-94). Significant decrease in phosphorylation of PKG-Iα at Ser50, Ser72, Ser89 was induced by HR injury while SCU treatment significantly increased the phosphorylation of PKG-Iα, not only at Ser50, Ser72 and Ser89, but also at Ser44 and Thr58 (two novel phosphorylation domains). Our results demonstrate PKG-Iα might play an important role in the protective effects of SCU on ED against MIR injury.