Abstract Body: Bone marrow mesenchymal stem cells (BMSCs) differentiate into various type of cells and secret a great number of bioactive factors, showing preliminary efficacy in the preclinical stroke models. However, low survival rate, limited homing and secreting capacity restricted the success application of BMSCs in stroke clinical trials. Pyruvate kinase M2 (PKM2), which has both metabolic enzymatic and protein kinase activity, reduced infarct volume and enhanced functional recovery in focal cerebral ischemia mice by promoting angiogenesis and neurogenesis. Hypoxia preconditioning can improve survival and increase the migration and secreting ability of BMSCs in vitro and in vivo. Moreover, under hypoxia circumstance, PKM2 translocated into the nuclear further activated the gene transcription including HIF-1α and pro-regenerative factors. We hypothesized that PKM2 overexpression combined with hypoxia preconditioning increased the bioactivity of BMSCs and investigated whether delayed intranasal delivery of BMSCs treated with PKM2 overexpression and hypoxia preconditioning could maximize neurological recovery after stroke. Our in vitro western blot results showed that PKM2 overexpression combined with hypoxia preconditioning upregulated the expression of HIF-1α and angiopoietin-1 (ANG-1) in BMSCs. At 21 days after stroke, BMSCs treated with PKM2 overexpression and hypoxia preconditioning increased the DiI perfused vessels than the PKM2-BMSC group or the HYPO-BMSC group. The PKM2-HYPO-BMSC group further exhibited higher cerebral perfusion unit and NeuN labeled cortical area than the PKM2-BMSC group and the HYPO-BMSC group. In gait analysis, mice received PKM2-HYPO-BMSC treatments displayed significant decreased step cycle time compared with other groups. Our data suggested that delayed intranasal delivery 3 of BMSCs treated with PKM2 overexpression and hypoxia preconditioning increased artery density, local cerebral blood flow (LCBF), functional recovery and reduced cortical tissue loss following ischemic stroke. These results highlighted BMSCs treated with PKM2 overexpression and hypoxia preconditioning delivery as an optimized strategy for ischemic stroke treatment.