Sex related differences in cardiovascular function have been reported in oestrogen receptor β knockout (ERβKO) mice. In this study we examined the role of endothelium‐derived hyperpolarizing factor (EDHF) in differences in small artery endothelial function between ERβKO and wild‐type (WT) mice. Small femoral arteries were isolated from ERβKO and WT mice and mounted on a wire myograph. Concentration–response curves to ACh were compared before and after incubation with inhibitors of nitric oxide (NO) and prostacyclin (PGI2) synthesis. Comparison of the expression of the principal vascular connexins (Cx37, 40 and 43), implicated in EDHF‐mediated dilatation were undertaken by immunohistochemistry. Vascular ultrastructure was studied by transmission and scanning electron microscopy. ACh‐induced relaxation of arteries (< 200 μm internal diameter) was greater in WT females versus males and was attributable to a greater EDHF component of relaxation. This sex difference was absent in ERβKO mice. Arteries from ERβKO males (but not females) were more sensitive to ACh compared to WT. The pharmacological evidence and morphological prerequisite for involvement of gap junctions in EDHF‐mediated responses was confirmed in male arteries. The absence of ERβ had no influence on expression of main Cx subtypes within vascular wall or on ultrastructure and morphology of the endothelium. The data suggest that in WT male mice, ERβ reduces EDHF‐mediated relaxation through gap junction communication.