Background and purpose: Ischemic stroke stands as a primary contributor to global disability and mortality, with treatment primarily focus on reducing neuronal death. Exosomes carry various miRNAs as a tool for intercellular communication and signal transduction, which provides a novel treatment for stroke. This research aims to reveal the protective effect and mechanisms of astrocyte-derived exosomes (AS-Exos) against ischemic stroke in mouse. Experimental Approach: AS-Exos were isolated and identified, and blood brain barrier (BBB) permeability and cellular localization were determined. MiR-664-5p overexpression (OE) or knockdown (KD) exosomes were constructed and extracted. In vitro, cell viability, lactate dehydrogenase (LDH) releasing, apoptosis and related factors of oxygen-glucose deprivation/reoxygenation (OGD/R) neurons were measured. In vivo, cerebral infarction volume, brain water content, BBB permeability, neurological score, inflammatory cytokines, and apoptosis factors were measured at 24 h after middle cerebral artery occlusion/reperfusion (MCAO/R). Cerebral blood flow (CBF), sensorimotor function, spatial learning and memory function, and pathological analysis were evaluated at 7 days after MCAO/R. Key Results: MiR-664-5p OE exosomes protected neuron by increasing cell viability, decreasing LDH release, inhibiting neuronal apoptosis after OGD/R injury. Furthermore, it alleviated ischemic stroke by reducing infarction volume and brain water content, sustaining BBB integrity, and inhibiting apoptosis. Increased CBF, improved neurological functional recovery and alleviated pathological damage were also observed. Meanwhile, miR-664-5p KD exosomes administration yielded the opposite results, aggravated ischemic brain injury. Conclusion and Implications: MiR-664-5p carried in astrocyte-derived exosomes alleviates experimental ischemic stroke via inhibiting neuronal apoptosis, which may be a new target for ischemic stroke treatment.