Whether the effect of miR-181a is sexually dimorphic in stroke is unknown. Prior work showed protection of male mice with miR-181a inhibition. Estrogen receptor-α (ERα) is an identified target of miR181 in endometrium. Therefore we investigated the separate and joint effects of miR-181a inhibition and 17β-estradiol (E2) replacement after ovariectomy. Adult female mice were ovariectomized and implanted with an E2- or vehicle-containing capsule for 14d prior to 1 h middle cerebral artery occlusion (MCAO). Each group received either miR-181a antagomir or mismatch control by intracerebroventricular injection 24 h before MCAO. After MCAO neurologic deficit and infarct volume were assessed. Primary male and female astrocyte cultures were subjected to glucose deprivation with miR-181a inhibitor or transfection control, and E2 or vehicle control, with/without ESRα knockdown with small interfering RNA. Cell death was assessed by propidium iodide staining, and lactate dehydrogenase assay. A miR-181a/ERα target site blocker (TSB), with/without miR-181a mimic, was used to confirm targeting of ERα by miR-181a in astrocytes. Individually, miR-181a inhibition or E2 decreased infarct volume and improved neurologic score in female mice, and protected male and female astrocyte cultures. Combined miR-181a inhibition plus E2 afforded greater protection of female mice and female astrocyte cultures, but not in male astrocyte cultures. MiR-181a inhibition only increased ERα levels in vivo and in female cultures, while ERα knockdown with siRNA increased cell death in both sexes. Treatment with ERα TSB was strongly protective in both sexes. In conclusion, the results of the present study suggest miR-181a inhibition enhances E2-mediated stroke protection in females in part by augmenting ERα production, a mechanism detected in female mice and female astrocytes. Sex differences were observed with combined miR-181a inhibition/E2 treatment, and miR-181a targeting of ERα.