Purpose: Cerebral ischemia/reperfusion injury (CIRI) severely impacts patient outcomes and quality of life, with limited treatment options. Although fibroblast growth factor21 (FGF21) is known for its metabolic and anti-inflammatory effects, its role and mechanisms in CIRI are not well explored. Methods: After developing an MCAO/R injury model, mice received intraperitoneal injections of FGF21 (1.5 mg/kg) 15 min prereperfusion, as well as 8 and 16 h post-reperfusion. The TTC, TUNEL, H&E, and Nissl stainings were used 24 h post-reperfusion to determine the infarct volume, apoptotic cells, brain pathological damage, and nerve cell survival, respectively. ELISA and Western blotting were employed to detect oxidative stress (OxS) products and ferroptosis-related markers. RNA-seq of the ischemic penumbra tissues was conducted, followed by bioinformatics analysis to screen and identify differentially expressed genes (DEGs). Then, we used qPCR to validate relevant molecule mRNA expression while using immunofluorescence staining to assess CYBB protein localization and expression. Results: The FGF21 reduced the infarct volume in MCAO/R-injured mice, diminished apoptotic cell numbers, and alleviated pathological damage to ischemic brain tissue. Furthermore, FGF21 inhibited OxS and ferroptosis post-CIRI. RNA-seq revealed a significant differential expression of numerous genes, extensively involving diverse biological processes post- ischemia/reperfusion injury (IRI). Bioinformatics analysis and validation results indicated that CYBB was the most significantly differentially expressed ferroptosis-related molecule, and it may be a novel key regulatory molecule mediating anti-IRI of FGF21. Conclusion: FGF21 protects CIRI by inhibiting OxS and ferroptosis. The CYBB, a new key regulator, may mediate its antiferroptotic effects, offering new insights into CIRI therapies.