We studied the roles of estrogen receptors (ER) and aromatase in the mediation of flow-induced dilation (FID) in isolated arteries of male ER knockout (ER-KO) and wild type (WT) mice. FID was comparable between gracilis arteries of WT and ER-KO mice. In WT arteries, inhibition of NO and prostaglandins eliminated FID. In ER-KO arteries, L-NAME inhibited FID by ~26%, whereas indomethacin inhibited dilations by ~50%. The remaining portion of the dilation was abolished by additional administration of PPOH or iberiotoxin, inhibitors of epoxyeicosatrienoic acid (EET) synthesis and large conductance potassium channels, respectively. By using an electrophysiologic technique, we found that in the presence of 10 dyne/cm² shear stress, the perfusate passing through donor vessels isolated from gracilis muscle of ER-KO mice subjected to L-NAME and indomethacin, elicited smooth muscle hyperpolarization and a dilator response of endothelium-denuded detector vessels. These responses were prevented by the presence of iberiotoxin in detector, or PPOH in donor vessels. GC-MS analysis indicated a significant increase in arterial production of EETs in ER-KO compared to WT mice. Western blotting showed a significantly reduced eNOS expression, but enhanced expressions of aromatase and ER in ER-KO arteries. Treatment of ER-KO arteries with specific aromatase siRNA for 72 hours, knocked down the aromatase mRNA and protein, that was associated with the elimination of EET-mediation of FID. Thus, FID in male ER-KO arteries is maintained via an endothelium derived hyperpolarizing factor/EET-mediated mechanism compensating for a reduced NO mediation, due, at least in part, to estrogen aromatized from testosterone.