Objective To investigate the mechanism of the effect of acetylglucosamine transferase 7 (GalNAc-T7) on the injury of H9c2 cardiomyocytes induced by hypoxia/reoxygenation under high glucose environment, and its regulatory mechanism on endoplasmic reticulum stress (ERS). Methods The cell experiments were divided into: control group (Control) cells, transfected with pc NC cells; HG group: 50 mmol/L glucose was added to the culture medium, and cells were transfected with pc-NC； HG+H/R group: Simultaneously with HG treatment, low oxygen culture was performed for 6 hours, and cells were transfected with pc-NC； Overexpression group (pc), cells were transfected with pc while undergoing HG+H/R treatment； ERS agonist, Tunicamycin (Tun) group: Cells were treated with HG+H/R and pc transfection, and 2 mg/L Tun was added to the cell culture medium. EdU staining was used to detect the proliferation activity of cells. AO staining was used to detect the apoptosis rate of cells. ER Tracker Red staining was used to detect endoplasmic reticulum damage in cells. Fluo-3/AM staining was used to detect the content of the calcium ion. DCFH-DA staining was used to detect the level of ROS in cells. Western blot was used to detect the expressions of GalNAc-T7, PERK, p-PERK, GRP78, eIF2α, p-eIF2α, ATF4 and CHOP in cells. Results Overexpression of GalNAc-T7 could significantly increase the proliferation activity of H9c2 cells in HG+H/R, reduce cell apoptosis rate, alleviate endoplasmic reticulum damage, downregulate the levels of calcium ions and ROS in cells, and reduce the expression levels of p-PERK/PERK, GRP78, p-eIF2α/eIF2α, ATF4, and CHOP. Tun could partially reverse the protective effect of overexpression of GalNAc-T7 on the cardiomyocytes, and the differences were statistically significant (all P＜0.05). Conclusion Overexpression of GalNAc-T7 could significantly inhibit ERS and alleviate H9c2 high glucose hypoxia/reoxygenation injury damage