Advanced glycation end products (AGEs) disturb endothelial barrier function and contribute to age-related diseases. As microRNAs (miRNAs) are potential therapeutic agents, targeting AGEs-associated signaling using miRNAs in endothelial cells may be an effective intervention strategy for age-related vascular disorders. This study investigated the effects of AGEs on the endothelial cell senescence and barrier function in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with AGEs and transfected with miRNA-1-3p mimics to induce overexpression of miR-1-3p. Senescence-associated β-galactosidase (SA-β-Gal) staining and senescence-related proteins P53, P21, and P16 were detected to evaluate the endothelial cell senescence. The expression levels of myosin light chain kinase (MLCK) signaling and transendothelial electric resistance (TEER) were used to indicate endothelial barrier function. AGEs significantly increased SA-β-gal staining-positive cells accompanied by the upregulation of P53, P21, and P16 expression. AGEs also damaged endothelial barrier function by decreasing TEER and increasing zonula occludens protein 1, p-MLC/MLC, and MLCK. miRNA-1-3p was significantly reduced in HUVECs treated with AGEs. miR-1-3p overexpression decreased MLCK signal and improved AGEs-induced endothelial barrier function impairment. Meanwhile, miR-1-3p overexpression ameliorated oxidative stress and endothelial cell senescence induced by AGEs. AGEs induced endothelial cell senescence and endothelial barrier dysfunction by regulating miR-1-3p/MLCK signaling pathway.Copyright © 2022 Elsevier B.V. All rights reserved.