The role of the estrogen receptor (ER) subtypes in the modulation of vascular function is poorly understood. The aim was to characterize ex-vivo the functional properties of small arteries and their response to estrogens in the mesenteric circulation of female and male estrogen receptor (ER) knock out mice (ERKO) and their wild type (WT) littermates. Responses to changes in intraluminal flow and pressure were obtained before and after incubation with 17-estradiol or ER agonist propyl-pyrazole-triol (PPT, 3h; 10nM). Cumulative concentration-response curves to acetylcholine (ACh), norepinephrine (NE) and passive distensibility were compared in respect to sex and genotype. The collagen and elastin content within the vascular wall and ERs expression were also determined. Endothelial morphology was visualized by scanning electron microscopy. 17-estradiol and PPT treated arteries from female ERKO and WT mice showed enhanced flow mediated dilation, but this was not evident in males. Distensibility was decreased in arteries from ERKO females. Sex differences in myogenic tone were observed in 17-estradiol treated arteries, but were similar between ERKO and WT mice. ACh- and NE-induced responses were similar between groups and sexes. ER was similarly expressed in the endothelium and media of arteries from all groups studied, as well as ER in WT animals. Endothelial morphology was similar in arteries from animals of both sexes and genotype, however arterial elastin content was decreased and collagen content was increased in ERKO male as compared to WT male and to ERKO female. We suggest that ERs play a sex- specific role in estrogen-mediated flow responses and distensibility, and that deletion of ER affects artery structure but only in male animals.