EDA2R is a member of the large family of tumor necrosis factor receptor (TNFR). Previous studies suggested that EDA2R expression might be increased in the kidneys of diabetic mice. However, its mRNA and protein expression in kidneys were not analyzed; moreover, its role in the development of diabetic kidney disease was not explored. Here we analyzed the mRNA and protein expressions of EDA2R in diabetic kidneys and examined its role in the podocyte injury in high glucose milieu. By analysis with real-time PCR, Western blotting, we found that both the mRNA and protein levels of EDA2R were increased in the kidneys of diabetic mice. Immunohistochemical studies revealed that EDA2R expression was enhanced in both glomerular and tubular cells of diabetic mice and humans. In vitro studies, high glucose increased EDA2R expression in cultured human podocytes. Overexpression of EDA2R in podocytes promoted podocyte apoptosis and decreased nephrin expression. Moreover, ED2AR increased ROS generation in podocytes, while inhibiting ROS generation attenuates EDA2R-mediated podocyte injury. In addition, EDA2R silencing partially suppressed high glucose-induced ROS generation, apoptosis, and nephrin decrease. Our study demonstrated that high glucose increases EDA2R expression in kidney cells and that EDA2R induces podocyte apoptosis and dedifferentiation in high glucose milieu partially through enhanced ROS generation. Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.