Mitochondria are important organelles that regulate adenosine triphosphate (ATP) production, intracellular calcium buffering, cell survival, and apoptosis. They play therapeutic roles in injured cells via transcellular transfer through extracellular vesicles, gap junctions, and tunneling nanotubes. Astrocytes secrete numerous factors that promote neuronal survival, synaptic formation, and plasticity. Recent studies have demonstrated that astrocytes transfer mitochondria to damaged neurons to enhance their viability and recovery. In this study, we observed that treatment with mitochondria isolated from rat primary astrocytes enhanced cell viability and ameliorated hydrogen peroxide-damaged neurons. Interestingly, isolated astrocytic mitochondria increased the number of cells in damaged neuronal conditions but not in normal conditions, although the mitochondrial transfer efficiency did not differ between the two conditions. This effect was also observed after astrocytic mitochondrial transplantation in a rat middle cerebral artery occlusion model. These findings suggest that mitochondria transfer therapy can be used to treat acute ischemic stroke and other diseases.