In the current investigation, we explored the benefits of aucubin against rodent ischemia/reperfusion (I/R) damages in brains and elucidated the role of 5'-AMP-activated protein kinase (AMPK) in its neuroprotective action. I/R model of brain was established in male three-month-old rats through 2 h of middle cerebral artery occlusion followed by two days of reperfusion. Aucubin boosted phosphorylation of AMPKα in ipsilateral cortex of injured rats. Then, rats were exposed to cerebral I/R damage and received treatment of aucubin and compound C (a well-known AMPK inhibitor). It was found that aucubin administration improved neurological symptom score, decreased infarct volume, and mitigated cerebral edema in injured rats. Aucubin administration upregulated Nrf2 expression and abated oxidative stress in ipsilateral cortex of injured rats. Aucubin administration reduced levels of multiple pro-inflammatory cytokines, suppressed microglial activation and neutrophil infiltration, and promoted M2 polarization in injured rats. More importantly, compound C abolished the neuroprotective, anti-oxidant and inflammation-modulating effects of aucubin in injured rats, at least in part. Therefore, we concluded that activation of AMPK by aucubin alleviated I/R injury in brain through abating oxidative stress and suppressing inflammation, identifying a potential candidate for those patients of ischemic stroke.© 2023. The Author(s), under exclusive licence to Cell Stress Society International.