BACKGROUND: We previously demonstrated that exercise pretreatment can suppress oxidative stress and neuroinflammation following ischemic stroke. However, the specific mechanisms underlying these effects are uncertain. Sestrin2 (Sesn2), a stress-responsive protein, has been reported to reduce neuroinflammation and protect against ischemic cerebral injury. Hence, this study aimed to verify whether Sesn2 can mediate the antineuroinflammatory and antioxidative effects of exercise pretreatment and explore the potential downstream mechanisms involved. METHODS: To assess infarction volume and neuronal morphology, we employed HE staining. Neurological functions following ischemic stroke were evaluated via modified neurological severity scores. Techniques such as immunofluorescence, TUNEL, Fluoro-Jade B, dihydroethidium staining, and Western blotting were utilized to investigate neuronal injury, oxidative stress, neuroinflammation, autophagic flux, and signaling pathway molecules. RESULTS: Our findings revealed that in a middle cerebral artery occlusion (MCAO) mouse model, administration of Sesn2 shRNA abolished the neuroprotective effects induced by exercise pretreatment. These effects include improvements in neurological dysfunction and impaired autophagy, as well as a reduction in oxidative stress and neuroinflammation. Mechanistically, the administration of AICAR to activate the AMPK/TFEB signaling pathway significantly reversed the aforementioned effects. Moreover, the inhibition of autophagic flux by chloroquine (CQ) in MCAO mice pretreated with exercise led to increased neuroinflammation. CONCLUSIONS: Sesn2 contributes to the positive outcomes of exercise pretreatment for ischemic stroke. Sesn2 exerts neuroprotection by inhibiting oxidative stress and neuroinflammation, potentially through AMPK/TFEB-mediated autophagic flux in MCAO. Sesn2 may hold promise as a novel exercise-mimetic molecule and a potential target for therapeutic interventions in ischemic stroke.