Objectives: Cerebral ischemia-reperfusion (I/R) injury is the leading cause of ischemic stroke. Pyruvate Kinase isozymes M2 (PKM2), as a critical glycolytic enzyme during glycolysis, is involved in neuronal apoptosis in rats with hypoxic-ischemic encephalopathy. This study focused on functional investigation and potential molecular mechanism toward PKM2 in cerebral I/R injury. Methods: Cerebral I/R injury model was established by middle cerebral artery occlusion (MCAO) in vivo or oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro. qRT-PCR and Western blot were used to detect the expression of PKM2 in I/R injury models. The effects of PKM2 on I/R injury were determined via triphenyl tetrazolium chloride staining and evaluation of neurological deficits. Cell Counting Kit-8 was employed to detect cell viability, and ELISA was conducted to detect pro-inflammatory cytokines. The underlying mechanism involved in regulation of PKM2 on I/R injury was investigated via ELISA and Western blot. Results: PKM2 was upregulated after cerebral I/R injury. Knockdown of PKM2 alleviated MCAO-induced infarction and neurological dysfunction. Moreover, PKM2 knockdown also alleviated OGD/R-induced neuronal cell injury and inflammatory response. Mechanistically, PKM2 knockdown-induced neuroprotection was accompanied by inhibition of high-mobility group box 1 (HMGB1), reflected by inactivation of TLR4/MyD88 (myeloid differentiation factor 88)/TRAF6 (TNF receptor-associated factor 6) signaling pathway. Conclusions: Knockdown of PKM2 attenuated cerebral I/R injury through HMGB1-mediated TLR4/MyD88/TRAF6 expression change, providing a potential target for cerebral I/R injury treatment.