BackgroundMicroglial polarization is one of the most promising therapeutic targets for multiple central nervous system (CNS) disorders, including ischemic stroke. However, detailed transcriptional alteration of microglia following cerebral ischemic stroke remains largely unclear.MethodsFocal cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) for 60 min in mice. Single-cell RNA sequencing (scRNA-seq) was performed using ischemic brain tissues from tMCAO and sham mice 3 days after surgery.Ch25h−/−mice were used to investigate the role of specific microglia subcluster on post-stroke infarct volume and neuroinflammation.ResultsWe identified a relatively homeostatic subcluster with enhanced antigen processing and three “ischemic stroke associated microglia” (ISAM): MKI67+, CH25H+and OASL+subclusters. We found the MKI67+subcluster undergo proliferation and differentiation into CH25H+and OASL+subclusters. CH25H+microglia was a critical subcluster of ISAM that exhibited increased phagocytosis and neuroprotective property after stroke.Ch25h−/−mice developed significantly increased infarct volume following ischemic stroke compared toCh25h+/−. Meanwhile, the OASL+subcluster accumulated in the ischemic brain and was associated with the evolving of neuroinflammation after stroke, which was further aggravated in the aged mice brain.ConclusionsOur data reveal previously unrecognized roles of the newly defined CH25H+and OASL+microglia subclusters following ischemic stroke, with novel insights for precise microglia modulation towards stroke therapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-023-02799-6.