〔Abstract〕 Objective To explore the effect of melatonin(MLT) on human umbilical vein endothelial cell(HUVEC) injury induced by high glucose and to explore its probable molecular mechanism. Methods HUVEC were cultured and divided into three groups: control group, model group and treatment group. The control group(Ctrl group) was treated with DMEM medium at a normal low glucose level(5.5 mmol/L glucose), and the model group(HG group) was treated with DMEM medium at a high glucose level(30 mmol/L glucose). Treatment group(HG+MLT group) was treated with DMEM medium at a high glucose level containing 100 μmol/L melatonin. The method of MTT was used to detect the effects of high glucose and melatonin on HUVEC proliferation. The release of lactate dehydrogenase(LDH) in cell culture supernatant was determined. The production of reactive oxygen species(ROS) was detected by flow cytometry. Hoechst 33342/PI double-staining was used to detect cell apoptosis and necrosis. Western blot was used to detect the effects of high glucose and melatonin on endoplasmic reticulum stress and cell pyroptosis related protein expression in HUVEC. Results Compared with the control group, cell proliferation level of the model group decreased, LDH releasing in cell culture supernatant increased, ROS production increased, the blue and red fluorescence increased in the double-staining, the expressions of GRP78, ATF4, CHOP, IRE1α, PERK, ATF6α, NLRP3, cleaved caspase-1, IL-1β increased. Compared with model group, cell proliferation level of the treatment group increased, LDH releasing in cell culture supernatant decreased, ROS production decreased, the blue and red fluorescence decreased in the double-staining, the expressions of GRP78, ATF4, CHOP, IRE1α, PERK, ATF6α, NLRP3, cleaved caspase-1, IL-1β decreased. Conclusion MLT can alleviate HUVEC damage induced by high glucose, and the probable mechanism may be related to the regulation of endoplasmic reticulum stress and cell pyroptosis.