Abstract: Objective To investigate the protective effect of astilbin on hydrogen peroxide (H₂O₂)-induced injury in human umbilical vein endothelial cells (HUVEC) and related mechnisms. Methods The experimental concentration of astilbin was screened by CCK8 assay. Endothelial cells were divided into four groups: control group, H₂O₂ group, and medium/high-concentration (50 and 100 μmol/L) astilbin groups. An oxidative injury model of endothelial cells was established by H₂O₂ induction at 400 μmol/L. Cell viability, malondialdehyde (MDA) content, activities of glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC) and superoxide dismutase (SOD), intracellular reactive oxygen species (ROS), and nitric oxide (NO) were measured. The mRNA expression of fas, caspase-8, Bax, Bcl-2, and caspase-3 was detected by real-time fluorescence quantitative PCR (RT-qPCR). The protein expression of Bax, Bcl-2, Cyto-C, p53, and cleaved-caspase-3 was measured by Western blot. Results CCK8 Results showed that astilbin exerted no toxicity to HUVEC within the concentration range of 1-100 μmol/L. Compared with the control group, the H₂O₂ group showed significantly reduced cell viability, with cell shrinkage, and presented increases in MDA content, decreases in GSH-Px, T-AOC and SOD activities, and increases in ROS accumulation and NO release. The H₂O₂ group also exhibited increased mRNA and protein levels of Cyto-C, Bax, p53, and caspase-3, and decreased mRNA and protein levels of Bcl-2. Compared with the H₂O₂ group, the astilbin group showed improved cell morphology, reduced MDA content, enhanced GSH-Px, T-AOC, and SOD activities, reduced ROS and NO release, and an increasing trend in Bcl-2 protein expression, and inhibited expression of Cyto-C, Bax, p53, and caspase-3. Conclusions Astilbin exert significant protective effect on H₂O₂-induced endothelial cell injury, which may be related to improved intracellular oxidative stress and apoptosis.