To explore risk factors for stroke independent of hypertension and the relationship between riboflavin kinase (RFK) and stroke. Gene expression profiling in the brains of spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHRSP) was comparatively analyzed by gene chips. The differentially expressed gene RFK was further verified by q-PCR and Western blot. The protective role of RFK-regulated flavins (including riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) in stroke was observed in middle cerebral artery occlusion (MCAO) mice. Influence of flavins on apoptosis and death in oxygen and glucose deprivation (OGD)-treated neurons was examined by flow cytometry. Bax and Bcl-2 proteins were detected by Western blot. Of the 76 differentially expressed genes, 41 genes were upregulated, and 35 genes were downregulated in SHRSP as compared with SHR. RFK was significantly downregulated in SHRSP. Flavins markedly decreased infarct area in MCAO mice, inhibited apoptosis and death in OGD-treated neurons, and decreased Bax protein expression. Physiological downregulation of RFK may be a new potential risk factor for stroke, which probably affects the absorbance and utility of riboflavin and further destroys the protective effect of flavins on stroke. RFK might act as a therapeutic target for stroke.