Expression of the 17β-estradiol (E2) synthesis enzyme aromatase is highly upregulated in astrocytes following brain injury. However, the precise role of astrocyte-derived E2 in the injured brain remains unclear. In the current study, we generated a glial fibrillary acidic protein (GFAP) promoter-driven aromatase knock-out (GFAP-ARO-KO) mouse model to deplete astrocyte-derived E2 in the brain and determine its roles after global cerebral ischemia (GCI) in male and female mice. GFAP-ARO-KO mice were viable and fertile, with normal gross brain structure, normal morphology, intensity and distribution of astrocytes, normal aromatase expression in neurons, and normal cognitive function basally. In contrast, after GCI, GFAP-ARO-KO mice: (1) lacked the normal elevation of astrocyte aromatase and hippocampal E2 levels; (2) had significantly attenuated reactive astrogliosis; and (3) displayed enhanced neuronal damage, microglia activation, and cognitive deficits. RNA-sequencing (RNA-seq) analysis revealed that the i...