Objective To investigate the mechanism of the fibroblast growth factor 21 ( FGF21) / β-klotho / fibroblast growth factor type 1 receptor (FGFR1) pathway in brain injury of type 2 diabetes mellitus (T2DM) model rats with focal cerebral ischemia. Methods SD rats were fed a high fat diet and injected intraperitoneally with streptozotocin (STZ) to establish the T2DM rat model. The middle cerebral artery occlusion ( MCAO) model was established by the suture method. Rats were divided into normal control group ( Control group), simple cerebral ischemia group ( MCAO group), T2DM cerebral ischemia group (T2DM + MCAO group), T2DM cerebral ischemia FGFR1 inhibitor PD173074 treatment group ( PD173074 group ) and T2DM cerebral ischemia FGFR1 agonist PF05231023 treatment group (PF05231023 group) with 12 rats in each group. PD173074 and PF05231023 groups were injected with 5 mg / kg PD173074 and 5 mg / kg PF05231023 respectively at 30 minutes before MCAO. After MCAO for 24 hours, the neurological deficit score was calculated and the rats were sacrificed to obtain brain tissue samples. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was used to detect neuronal apoptosis. Real-time quantitative PCR was used to detect mRNA expression of FGF21, β-klotho and FGFR1. Protein expression of FGF21, β-klotho, FGFR1, platelet endothelial cell adhesion molecule (CD31), endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) was detected by Western blot. Results Compared with the control group, the neurological deficit score, apoptosis rate of nerve cells, mRNA and protein levels of FGF21, β-klotho, FGFR1 and protein levels CD31, ET-1, VEGF were significantly increased in MCAO and T2DM + MCAO groups (P<0. 05). Compared with the T2DM + MCAO group, the neurological deficit score, apoptosis rate of nerve cells, protein levels of CD31, ET-1 and VEGF were increased in the PD173074 group, while the mRNA and protein levels of FGF21, β-klotho and FGFR1 were decreased (P< 0. 05). Moreover, the neurological deficit score, apoptosis rate of nerve cells and the protein levels of CD31, ET-1 and VEGF in the PF05231023 group were decreased, while the mRNA and protein levels of FGF21, β-klotho and FGFR1 were increased (P< 0. 05). Conclusions Activation of the FGF21 / β-klotho / FGFR1 pathway may play an important protective role in focal cerebral ischemia model rats with T2DM.