As the resident immune cells in the central nervous system, microglia exhibit a 'sensitized' or 'primed' phenotype with dystrophic morphology and dysregulated functions in aged brains. Although studies have demonstrated the inflammatory profile of aged microglia in several neurological diseases, this issue is largely uncertain in stroke. Consequently, this study investigated the effects of primed and repopulated microglia on post-ischemic brain injury in aged mice. We replaced primed microglia with newly repopulated microglia through pharmacological administration and withdrawal of the colony-stimulating factor 1 receptor (CSF1R) inhibitor, PLX3397. Further, we performed a series of behavioral tests and flow cytometry in mouse models of middle cerebral artery occlusion (MCAO) to study the effects of microglial replacement on ischemic injury in the aged brain. With depletion and subsequent repopulation of microglia in MCAO mice, microglial replacement in aged mice improved neurological function and decreased brain infarction. This protective effect was accompanied by the reduction of peripheral immune cells infiltrating into brains. We showed that the repopulated microglia expressed elevated neuroprotective factors (including Cluster of Differentiation 206, transforming growth factor-β, and interleukin-10) and diminished expression of inflammatory markers (including Cluster of Differentiation 86, interleukin-6, and tumor necrosis factor α). Moreover, microglial replacement protected the blood-brain barrier and relieved neuronal death in aged mice subjected to 60 min of MCAO. These results imply that the replacement of microglia in the aged brain may alleviate brain damage and neuroinflammation, and therefore, ischemic brain damage. Thus, targeting microglia could be a promising therapeutic strategy for ischemic stroke.