Stroke is one of the most fatal diseases in the world, which is seriously threatening human life. Raf kinase inhibitory protein (RKIP) is involved in the regulation of several signaling pathways and is important for cell growth, proliferation, differentiation and apoptosis. In the present study, the protective effect of RKIP on stroke was investigated by the metabonomics method based on the UHPLC-Q-TOF-MS technique. TTC staining of brain tissues showed that RKIP overexpression by the lentivirus markedly reduced the necrotic area after ischemic stroke. Subsequent metabolomic profiling revealed that the protective effect of RKIP overexpression on ischemic stroke is mainly reflected in the metabolism of energy, amino acids and lipids. Several metabolites involved in purine, pyrimidine and fatty acid metabolism were identified. It was also shown that the protective effect of RKIP on ischemic stroke might be mediated by inhibiting the inflammatory response. The current study provided insight into the molecular mechanism of ischemic stroke and a reliable basis for the development of novel therapeutic targets for the treatment of ischemic stroke.