The current study aimed to explore the role of autophagy in cerebral ischemia-reperfusion injuries (CIRI) and elucidate the efficacy of liensinine treatment. An in vitro ischemia-reperfusion (I/R) neuronal cell model was established and pretreated with liensinine or rapamycin (RAPA). Cell proliferation and survival were detected using a cell counting kit-8 (CCK-8) assay, while cell damage and apoptosis were detected using the lactate dehydrogenase (LDH) leakage rate and flow cytometry. Autophagy activity was detected using monodansylcadaverine (MDC) staining. Thereafter, I/R models were established in vivo in rats and the presence of neurological deficits was examined. Hematoxylin-eosin (HE) and triphenyl tetrazolium chloride (TTC) staining was used to detect pathological damage in brain tissue and the volume ratio of the cerebral infarction. The levels of PI3K/AKT pathway-related proteins and autophagy-related proteins (mTOR, LC3, P62, and TSC2) were detected using Western blot. The findings showed that liensinine treatment increased cell viability, decreased cell injury and apoptosis, and inhibited autophagy. The addition of RAPA to promote autophagy inhibited cell viability and enhanced cell injury and apoptosis. The I/R rats in the model group exhibited deficient neurological function, while those in the liensinine treatment group showed restoration of normal neural function and reduction of the necrotic area and infarct volume ratio in the brain tissue. Furthermore, liensinine treatment also inhibited the PI3K/Akt pathway activity and autophagy. However, addition of RAPA reversed the effects of liensinine treatment and aggravated brain tissue injury. Therefore, liensinine can play a neuroprotective role in CIRI by inhibiting autophagy through regulation of the PI3K/Akt pathway.