The study investigates the role of histone deacetylase 3 (HDAC3) in microglial proliferation and polarization after ischemic stroke. Microglial HDAC3 knockout (HDAC3-miKO) mice showed improved long-term functional and histological outcomes post-stroke. RNA-seq analysis revealed that HDAC3-deficient microglia arrested mitosis, specifically inhibiting the proliferation of proinflammatory microglia without affecting anti-inflammatory microglia. ATAC-seq further showed that HDAC3-miKO induced the closure of accessible regions enriched with PU.1 motifs, suggesting PU.1 as a downstream molecule mediating HDAC3's effects. These findings highlight the HDAC3/PU.1 axis's role in regulating microglial proliferation and polarization, contributing to the pathophysiology of ischemic stroke.The study investigates the role of histone deacetylase 3 (HDAC3) in microglial proliferation and polarization after ischemic stroke. Microglial HDAC3 knockout (HDAC3-miKO) mice showed improved long-term functional and histological outcomes post-stroke. RNA-seq analysis revealed that HDAC3-deficient microglia arrested mitosis, specifically inhibiting the proliferation of proinflammatory microglia without affecting anti-inflammatory microglia. ATAC-seq further showed that HDAC3-miKO induced the closure of accessible regions enriched with PU.1 motifs, suggesting PU.1 as a downstream molecule mediating HDAC3's effects. These findings highlight the HDAC3/PU.1 axis's role in regulating microglial proliferation and polarization, contributing to the pathophysiology of ischemic stroke.