Different kinds of factors contribute to gastric ulcer development which characterized by damaging gastric mucosal layer. However, gastric vascular homeostasis is not well defined and whether andrographolide has a protective function is largely unknown. The goal of this study is to investigate the potential function roles and underlying mechanism by which andrographolide regulates gastric vascular homeostasis in vivo and in vitro. Gastric ulcer animal model induced on andrographolide pretreated C57/BL6 mouse by ethanol intragastric administration. Hematoxylin and Eosin Stain, Masson's trichrome stain and Immunohistochemistry stain performed to observe gastric vascular homeostasis, which associated hemorrhage, extracellular matrix deposition and macrophage infiltration. The activity of vascular endothelial cells were associated with the proliferation and migration, which were detected using cell counting, MTS, and wound scratch healing assay. The underlying endothelial glycolytic mechanism investigated in vivo and in vitro. Andrographolide pretreatment dramatically attenuates ethanol intragastric administration induced imbalance of gastric vascular homeostasis which characterized by severe hemorrhage, increase extracellular matrix deposition and augment macrophage infiltration. Andrographolide treatment conspicuous inhibits HUVEC-C activity characterized by suppressing proliferation and migration of endothelial cells. Mechanically, andrographolide treatment significant suppresses the expression of glycolytic genes, especially decrease PFKFB3 expression. The treatment with PFKFB3 inhibitor, 3-PO, exacerbates the inhibitory function of andrographolide on vascular endothelial cell proliferation and migration. Those data Suggests that andrographolide contributes to maintain gastric vascular homeostasis, at least partially, by inhibiting PFKFB3 mediated glycolysis pathway. Andrographolide plays a crucial role in maintaining gastric vascular homeostasis during gastric ulcer development through regulating vascular endothelial cell glycolytic pathway.