This study aimed to evaluate the effect of hyperbaric oxygen preconditioning (HBO2P) on ischemic metabolites and the glutamate level after permanent middle cerebral artery occlusion (MCAO). HBO2P was administered: five treatments, one treatment per day. The permanent rabbit MCAO models were induced by a modified transorbital approach. The microdialysis procedures were performanced in the right peri-infarct area and contralateral area. MCAO decreased glucose levels while increasing lactate, pyruvate and lactate/pyruvate ratios. Early increase of glycerol and glutamate were also shown. HBO2P stabilized the glucose level and decreased the lactate/pyruvate ratios and glycerol in the peri-infarct area. In addition, it inhibited the increase of the glutamate level. Our study demonstrated that MCAO led to the imbalance of brain energy metabolites and excitatory amino acids. The modulation of energy metabolism and glutamate may be one of the factors contributing to the neuroprotective property of HBO2P.