Estrogen determines sex differences in adrenergic vessel tone by regulation of endothelial β-adrenoceptor expression. Am J Physiol Heart Circ Physiol 317: H243–H254, 2019. First published May 31, 2019; doi:10.1152/ajpheart.00456.2018.— Vessels of female rats constrict less and relax more to adrenergic stimulation than vessels of males. Although we have reported that these sex-specific differences rely on endothelial β-adrenoceptors, the role of sex hormones in β-adrenoceptor expression and related vessel tone regulation is unknown. We investigated the role of estrogen, progesterone and testosterone on β-adrenoceptor expression and adrenergic vessel tone regulation, along with sex-specific differences in human mammary arteries. The sex-specific differences in vasoconstriction and vasorelaxation in rat vessels were eliminated after ovariectomy in females. Ovariectomy increased vessel vasoconstriction to norepinephrine more than twofold. Vasorelaxations by isoprenaline and a β₃-agonist were reduced after ovariectomy. Estrogen, but not progesterone substitution, restored sex-specific differences in vasoconstriction and vasorelaxation. Vascular mRNA levels of β₁-and β₃-but not β₂-adrenoreceptors were higher in vessels of females compared with males. Ovariectomy reduced these differences by decreasing β₁-and β₃-but not β₂-adrenoreceptor expression in females. Consistently, estrogen substitution restored β₁-and β₃-adrenoreceptor expression. Orchiectomy or testosterone treatment affected neither vasoconstriction and vasorelaxation nor β-adrenoceptor expression in vessels of male rats. In human mammary arteries, sex-specific differences in vasoconstriction and vasorelaxation were reduced after removal of endothelium or treatment with L-NMMA. Vessels of women showed higher levels of β₁-and β₃-adrenoceptors than in men. In conclusion, the sex-specific differences in vasoconstriction and vasorelaxation are common for rat and human vessels. In rats, these differences are estrogen but not testosterone or progesterone dependent. Estrogen determines these differences via regulation of vascular endothelial β₁-and β₃-adrenoreceptor expression. NEW & NOTEWORTHY This study proposes a mechanistic concept regulating sex-specific differences in adrenergic vasoconstriction and vasorelaxation. Estrogen increases vascular β₁-and β₃-adreno-ceptor expression in female rats. This and our previous studies demonstrate that these receptors are located primarily on endothelium and when activated by norepinephrine act via nitric oxide (NO). Therefore, β-adrenergic stimulation leads to a more pronounced vasorelaxation in females. Coactivation of endothelial β₁-and β₃-adrenoreceptors leads to higher NO release in vessels of females, ultimately blunting vasoconstriction triggered by activation of smooth muscle α-adrenoceptors.