The classification of microglial M1/M2 polarization in the acute phase of ischemic stroke remains controversial, which has limited further advances in neuroprotective strategy. To thoroughly assess the microglial phenotypes, we made the middle cerebral artery occlusion model in mice to simulate the acute pathological processes of ischemic stroke from normal conditions to acute cerebral ischemia and then to the early reperfusion period. The temporal changes in gene profiles, cell subtypes, and microglial function were comprehensively analyzed using single-cell RNA sequencing. We identified 37,614 microglial cells and divided them into eight distinct subpopulations. Mic_home, Mic_pre1, and Mic_pre2 subpopulations were three clusters mainly composed of cells from the control samples, in which Mic_home was a homeostatic subpopulation characterized by high expression of Hpgd and Tagap, and Mic_pre1 and Mic_pre2 were two clusters with preliminary inflammatory activation characteristics marked by P2ry13 and Wsb1 respectively. Mic_M1L1 and Mic_M1L2 subpopulations exhibited M1-like polarization manifested by the upregulation of inflammatory genes after ischemic stroke, while the intrinsic heterogeneity on the level of inflammatory responses and neurotrophic support properties was observed. Moreover, we identified three unique clusters of cells with low inflammation levels. Mic_np1, Mic_np2, and Mic_np3 were characterized by high expression of Arhgap45, Rgs10, and Pkm respectively. However, these cells did not show significant M2-like characteristics and their classic microglia function was also attenuated. These subpopulations exhibited higher activation of neuropeptide functional pathways. At last, we performed cell-cell communication analysis and identified major couplings contributing to the interaction between microglia and other cell populations. In summary, our study elucidated the temporal heterogeneity of microglia in the acute phase of ischemic stroke, which may facilitate the identification of effective neuroprotective targets to curb ischemic damage at an early stage.