Background: Hypoxia/reoxygenation (H/R) in human umbilical vein endothelial cells (HUVECs) induces oxidative stress and eventually leads to vascular injury. Objective: The aim of this study was to examine the effect ofmelatonin on HUVECs injured by H/R and explore the underlying mechanisms. Materials and Methods: A model of HUVECs under hypoxia/reoxygenation was established. Cell migration and adhesive ability was measured by wound healing and adhesion assays. Cell proliferation was measured by EdU assay. Production of reactive oxygen species (ROS) was evaluated by CM-H2DCFDA staining. Actin cytoskeleton rearrangement was examined by immunofluorescence.Western blotting analysis were used to analyze P38 and HSP27 phosphorylation levels. Results: H/R inhibited HUVEC proliferation, cell migratory and adhesive capacities, whereas melatonin (1~100 M) inhibited these effects in a dose-dependent manner. Melatonin alone did not affect HUVEC viability, however, it inhibited the increase in ROS production and cytoskeleton disruption elicited by H/R, and it dose-dependently prevented H/R-induced upregulation of P38 and HSP27 phosphorylation. In addition, the ROS scavenger N-acetyl- L-cysteine markedly inhibited increased phosphorylation levels of P38 and HSP27 under H/R. Conclusions: Melatonin may have a potential clinical effect in trials of H/R-induced vascular injury through its antioxidant property.