The incidence of ischemic stroke is significantly increased in postmenopausal women. However, the neuroprotective effects of estrogen replacement therapy (ERT) against stroke remain controversial, and the role of astrocytes in ERT has rarely been explored. In this study, we investigated the effects of estrogen and selective estrogen receptor (ER) agonists on astrocytes activation and neuronal apoptosis in mice under conditions of cell culture oxygen and glucose deprivation and reperfusion (OGD-R), and global cerebral ischemia (GCI). We demonstrated that hippocampal astrocytes primarily express ERβ. In astrocytes, 2.5-20 nM 17β-estradiol (E2) or 10 nM DPN (ERβ agonist) not 10 nM PPT (ERα agonist), significantly increased GFAP expression. And 10 nM E2, DPN or E2+MPP (ERα antagonist), but not PPT or E2+PHTPP (ERβ antagonist), significantly reduced neuronal apoptosis following the subjection of astrocyte and neuronal cocultures to OGD-R. We also found that either 50 μg/kg E2 or 8 mg/kg DPN replacement (3 weeks) significantly increased GFAP expression and reduced GCI-induced neuronal apoptosis in hippocampal CA1 region of ovariectomized mice. These results indicate that estrogen-induced neuroprotection against ischemia-reperfusion injury involves activation of astrocytes via ERβ. Thus, the discovery and design of astrocyte-selective ERβ modulators may offer a new strategy for ERT of ischemic stroke.