Cells universally adapt to ischemic conditions by turning on a transcription factor hypoxia-inducible factor (HIF), in which its role is known to differ widely across many different types of cells. Given that microglia have been reported as an essential mediator of neuroinflammation in many brain diseases, we examined the role of HIF in microglia in the progression of an acute phase of ischemic stroke by challenging our novel strains of myeloid-specific Hif-1 alpha or Hif-2 alpha knockout (KO) mice created by Cre-loxP system via middle cerebral artery occlusion (MCAO). We observed that Hif-1 alpha but not Hif-2 alpha KO mice exhibited an improved recovery compared to wild-type (WT) mice determined by behavioral tests. Immunostaining analyses revealed that there were increased numbers of both mature and immature neurons while microglia and apoptotic cells were significantly decreased in the dentate gyrus of Hif-1 alpha KO mice following MCAO. By isolating microglia with fluorescence-activated cell sorter, we found that HIF-1 alpha-deficient microglia were impaired in phagocytosis, reactive oxygen species (ROS) production, and tumor necrosis factor-alpha (TNF-alpha) secretion. We further observed a significant decrease in the expression of Cd36 and milk fat globule-epidermal growth factor 8 (Mfg-e8) genes, both of which contain hypoxia-responsive element (HRE). Knocking down either of these genes in BV2 microglial cells was sufficient to abrogate HIF-mediated increase in phagocytosis, production of intracellular ROS, or TNF-alpha secretion. Our results therefore suggest that HIF-1 alpha in microglia is a novel therapeutic target to protect neuronal survival following an acute phase of ischemic stroke.