Brain ischemia and reperfusion (I/R) injury occurs in various pathological conditions, but there is no effective treatment currently available in clinical practice. Methylene blue (MB) is a century-old drug with a newly discovered protective function in the ischemic stroke model. In the current investigation we studied the MB-induced neuroprotective mechanism focusing on stabilization and activation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) in an in vitro oxygen and glucose deprivation (OGD)-reoxygenation model. Methods: HT22 cells were exposed to OGD (0.1% O-2, 6 h) and reoxygenation (21% O-2, 24 h). Cell viability was determined with the calcein AM assay. The dynamic change of intracellular O-2 concentration was monitored by fluorescence lifetime imaging microscopy (FLTIM). Glucose uptake was quantified using the 2-[N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino]-2-Deoxy-D-Glucose (2-NBDG) assay. ATP concentration and glycolytic enzyme activity were examined by spectrophotometry. Protein content changes were measured by immunoblot: HIF-1 alpha, prolyl hydroxylase 2 (PHD2), erythropoietin (EPO), Akt, mTOR, and PIP5K. The contribution of HIF-1 alpha activation in the MB-induced neuroprotective mechanism was confirmed by blocking HIF-1 alpha activation with 2-methoxyestradiol-2 (2-MeOE2) and by transiently transfecting constitutively active HIF-1 alpha. Results: MB increases cell viability by about 50% vs. OGD control. Compared to the corresponding control, MB increases intracellular O-2 concentration and glucose uptake as well as the activities of hexokinase and G-6-PDH, and ATP concentration. MB activates the EPO signaling pathway with a corresponding increase in HIF-1 alpha. Phosphorylation of Akt was significantly increased with MB treatment followed by activation of the mTOR pathway. Importantly, we observed, MB increased nuclear translocation of HIF-1 alpha vs. control (about three folds), which was shown by a ratio of nuclear: cytoplasmic HIF-1 alpha protein content. Conclusion: We conclude that MB protects the hippocampus- derived neuronal cells against OGD-reoxygenation injury by enhancing energy metabolism and increasing HIF-1 alpha protein content accompanied by an activation of the EPO signaling pathway. (C) 2015 IBRO. Published by Elsevier Ltd. All rights reserved.