Ischemic stroke initiated by transient or permanent cerebral blood flow decline remains the leading cause of permanent disability in industrialized nations. Therapeutic strategies to improve patient recovery are remain limited. Hypoxia post-conditioning (HPostC) has been known to be neuroprotective against ischemic injuries in vivo and in vitro. Understanding its mechanism of action may promote its clinical translation. In this study, we devised a method of HPostC treatment to provide protection from a focal cerebral ischemic induced injury and to explore the underling mechanism. We found that our HPostC method improved energy supply by elevating the level of glucose, pyruvate and ATP/ADP ratio within the cerebral hemisphere in mice. In the distal middle cerebral artery occlusion (dMCAO) mice, this HPostC treatment reduced infarct size, and was associated with increased levels of pyruvate, pyruvate/lactate ratio and ATP/ADP ratio. Western blot analysis indicated that the HPostC treatment up-regulated AMPK signaling activities in the cerebral hemisphere. Our results suggest that this HPostC treatment exerts its neuroprotective effect by promoting glycolysis to elevate the ATP/ADP level, and the AMPK/PFKFB3 signaling pathway. These findings may provide biomarkers for clinical use of HPostC methods. Copyright © 2020. Published by Elsevier B.V.