Ischemic stroke is a severe neurodegenerative disease with a high mortality rate. Retinoic acid is a representative metabolite of vitamin A. It has many beneficial effects including anti-inflammatory, anti-apoptotic, and neuroprotective effects. The purpose of this study is to identify specific proteins that are regulated by retinoic acid in ischemic stroke. Middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemia. Retinoic acid (5 mg/kg) or vehicle was injected intraperitoneally into male rats for four days prior to MCAO operation. Neurobehavioral tests were performed 24 hr after MCAO and the cerebral cortex was collected for proteomic study. Retinoic acid alleviates neurobehavioral deficits and histopathological changes caused by MCAO. Furthermore, we identified various proteins that were altered by retinoic acid in MCAO damage. Among these identified proteins, adenosylhomocysteinase, isocitrate dehydrogenase [NAD] subunit α, glycerol-3-phosphate dehydrogenase, Rab GDP dissociation inhibitor β, and apolipoprotein A1 were down-regulated in MCAO animals with vehicle treatment, whereas retinoic acid treatment alleviated these reductions. However, heat shock protein 60 was up-regulated in MCAO animals with vehicle, while retinoic acid treatment attenuated this increase. The changes in these expressions were confirmed by reverse transcription-PCR. These proteins regulate cell metabolism and mediate stress responses. Our results demonstrated that retinoic acid attenuates the neuronal damage by MCAO and regulates the various protein expressions that are involved in the survival of cells. Thus, we can suggest that retinoic acid exerts neuroprotective effects on focal cerebral ischemia by modulation of specific proteins.