ETHNOPHARMACOLOGICAL RELEVANCE: Piperine (PIP), a main active component isolated from Piper nigrum L., exerts neuroprotective effects in a rat model of ischemic stroke (IS). However, studies on the effects of PIP on neuroprotection and autophagy after IS are limited. AIM OF THE STUDY: This study aimed to prove the protective effects of PIP against brain IS and elucidate its underlying mechanisms. MATERIALS AND METHODS: Specific pathogen-free male Sprague-Dawley rats were selected to establish a permanent middle cerebral artery occlusion model. The experiment was randomly divided into six groups: sham group, model group, PIP intervention group (10, 20, and 30 mg/kg group), and nimodipine group (Nimo group, 12 mg/kg). Neurological function score, postural reflex score, body swing score, balance beam test, and grip strength test were used to detect behavioral changes of rats. The area of cerebral infarction was detected by TTC staining, and the number and morphological changes of neurons were observed by Nissl and HE staining. In addition, the ultrastructure of hippocampal dentate gyrus neurons was observed using a transmission electron microscope. Western blot was used to detect the expression of PI3K/AKT/mTOR signaling pathway proteins and autophagy-related proteins, namely, Beclin1 and LC3, in the hippocampus and cortex. Cell experiments established an in vitro model of oxygen-glucose deprivation (OGD) with the HT22 cell line to verify the mechanism. The experiment was divided into five groups: control group, OGD group, OGD + PIP 20 μg/mL group, OGD + PIP 30 μg/mL group, and OGD + PIP 40 μg/mL group. CCK-8 was used to measure cell activity, and Western blot was used to measure the expression of PI3K/AKT/mTOR signaling pathway proteins and autophagy-related proteins (Beclin1 and LC3). RESULTS: Compared with the model group, the neurological function scores, body swing scores, and postural reflex scores of rats in the 10, 20, and 30 mg/kg PIP intervention groups and Nimo groups decreased, whereas the balance beam score and grip test scores increased (all p < 0.05). After 10, 20, and 30 mg/kg PIP and Nimo intervention, the cerebral infarction area of pMCAO rats was reduced (p < 0.01), and Nissl and HE staining results showed that the number of neurons survived in the 30 mg/kg PIP and Nimo intervention groups increased. Cell morphology and structure were significantly improved (p < 0.05). Most of the hippocampal dentate gyrus neurons and their organelles gradually returned to normal in the 30 mg/kg PIP and Nimo intervention groups, with less neuronal damage. The expression levels of p-mTOR, p-AKT, and p-PI3K in the hippocampus and cortex of the 30 mg/kg PIP and Nimo intervention groups decreased, whereas the expression level of PI3K increased (all p < 0.05). In addition, the expression level of autophagy-related proteins, namely, Beclin1 and LC3-II, in the 30 mg/kg PIP and Nimo intervention groups decreased (all p < 0.05). Results of CCK-8 showed that after 1 h of OGD, the 30 and 40 μg/mL PIP intervention groups had higher cell viability than the OGD group (p < 0.01). Western blot results showed that compared with the OGD group, the expression level of p-mTOR, p-AKT, and p-PI3K in the 30 and 40 μg/mL PIP intervention groups decreased, and the expression level of PI3K increased (all p < 0.05). Moreover, the expression level of autophagy-related proteins, namely, Beclin1 and LC3-II, in the 30 and 40 μg/mL PIP intervention groups decreased (all p < 0.05). CONCLUSIONS: This study shows that PIP is a potential compound with neuroprotective effects. PIP can inhibit the PI3K/AKT/mTOR pathway and autophagy. Its inhibition of autophagy is possibly related to modulating the PI3K/AKT/mTOR pathway. These findings provide new insights into the use of PIP for the treatment of IS and its underlying mechanism.