Objective: Detection of central nervous system (CNS) molecular defects in an animal model of multiple sclerosis. Materials and Methods: Experimental autoimmune encephalomyelitis (EAE) was induced by a myelin oligodendrocyte glycoprotein. Protein expression profiles in the central nervous system between healthy clinical scores 1 and 3 of EAE were studied using a two dimensional electrophoresis based proteomics approach coupled with MALDI TOF/TOF mass spectrometry. Results: We identified 8 mitochondrial proteins that were differentially expressed in CNS, all of them down-regulated in scores 1 and/or 3. Of these, 5 proteins belong to the mitochondrial respiratory chain including: NADH dehydrogenase (ubiquinone) Fe-S protein 8, cytochrome c oxidase Va, cytochrome c oxidase Vb, ATP5B, NADH dehydrogenase (ubiquinone) flavoprotein 2. We also observed down-regulation of three other mitochondrial proteins including: glutaredoxin 5, estradiol 17 beta-dehydrogenase 8 and isocitrate dehydrogenase. Conclusion: Down-regulation of mitochondrial proteins supported the hypothesis that hypoxia-like tissue injury in multiple sclerosis (MS) lesions may be due to mitochondrial impairment.