The endothelin system plays an important role in mediating vascular function. The endothelin-B receptor (ETBR) on endothelial cells mediates vasodilation via nitric oxide production. The vasodilatory effect of the ETBR is lost following menopause and may contribute to impaired vascular endothelial function in postmenopausal women (PMW). However, it is unclear if these functional changes are due to changes in ETBR expression on the endothelium. Therefore, the purpose of this study was to test the hypothesis that endothelial cell ETBR expression is lower in PMW compared with young women (YW). Primary endothelial cells were harvested from the antecubital vein of healthy PMW (n = 15, 60 ± 6 yr) and YW (n = 15, 22 ± 2 yr). Cells were identified as endothelial cells by staining for vascular endothelial cadherin, and nuclear integrity was assessed using 4',6-diamidino-2-phenylindole (DAPI). Within those cells, ETBR was quantified using immunocytochemistry; fluorescence intensity was measured in 30 cells and averaged for each participant. Endothelial function was assessed using brachial artery flow-mediated dilation (FMD). Endothelial cell ETBR expression was lower in PMW [0.46 ± 0.11 arbitrary units (AU)] compared with YW (0.58 ± 0.14 AU; P = 0.02). Furthermore, significant correlations between ETBR expression and FMD (r = 0.47, P < 0.01), total cholesterol (r = -0.38, P = 0.04), and LDL cholesterol (r = -0.39, P = 0.03) were observed. These data demonstrate that endothelial cell ETBR expression is attenuated in PMW. These novel findings provide additional insight into the mechanisms underlying vascular endothelial dysfunction in PMW.NEW & NOTEWORTHY Our study provides novel data demonstrating attenuated endothelial ETBR expression in postmenopausal women. Furthermore, our data extend current knowledge by demonstrating a positive relation between ETBR expression and brachial artery flow-mediated dilation. These findings provide additional mechanistic insight into vascular endothelial dysfunction in postmenopausal women.