Ischemic stroke is a leading cause of death and neurological disability though mechanisms involving oxidative stress and the activation of apoptosis-related pathways. Retinoic acid, an active metabolite of vitamin A, is known for its neuroprotective effects via antioxidant and anti-apoptotic mechanisms. 14-3-3 γ is abundantly expressed in the brain and plays a critical role in maintaining neuronal function and survival. However, the effect of retinoic acid on the expression of 14-3-3 γ protein has not been fully elucidated. The aim of this study was to determine whether retinoic acid regulates the expression of 14-3-3 γ protein in the cerebral cortex of a stroke animal model. Male rats were randomly divided into four groups: phosphate-buffered saline (PBS) + sham, retinoic acid + sham, PBS + middle cerebral artery occlusion (MCAO), and retinoic acid + MCAO. Focal cerebral ischemia was induced using the MCAO method. Retinoic acid (5 mg/kg) or PBS was administered intraperitoneally immediately after MCAO. Neurological deficit scores and corner tests were conducted 24 h after MCAO surgery, and the cerebral cortex was harvested for analysis of 14-3-3 γ expression at the mRNA and protein levels. Neurological assessments showed that retinoic acid treatment significantly alleviated MCAO-induced behavioral impairments. Proteomic analysis revealed that MCAO markedly reduced the expression of 14-3-3 γ protein in the cerebral cortex, whereas retinoic acid administration effectively attenuated this reduction. These findings were further supported by reverse transcription-PCR and Western blot analyses, which showed consistent results at both the mRNA and protein levels. Retinoic acid attenuates the ischemia-induced downregulation of 14-3-3 γ expression in the cerebral cortex. Our findings can suggest that retinoic acid exerts neuroprotective effects in ischemic brain injury through the regulation of 14-3-3 γ protein expression.