BACKGROUND AND AIMS: Smoking is a known risk factor for stroke. However, the 'stroke paradox' refers to the observation that stroke patients who smoke often have higher survival rates and better outcomes compared to non-smokers. In this sense, several studies have demonstrated that nicotine (3-[(2S)-1-methylpyrrolidin-2-yl]pyridine) exerts neuroprotective effects. Despite this, the molecular underpinnings of this phenomenon remain unclear. AMP-activated protein kinase (AMPK) is known to play a complex and controversial role in ischemic stroke, with recent evidence suggesting that AMPK inhibition has neuroprotective effects in acute ischemic injury. Nicotine has been shown to influence AMPK signaling in the brain, suppressing appetite and promoting brown fat thermogenesis via hypothalamic AMPK inhibition. Therefore, we hypothesized that the neuroprotective effect of nicotine in ischemia is due to its inhibitory action on AMPK. The aim of this study has been to investigate whether i) AMPK is involved in nicotine's neuroprotective effects on cerebral ischemia and ii) small extracellular vesicle (sEV)-mediated genetic inhibition of AMPK could replicate this effect in rodent models. METHODS: Male adult mice or rats subjected to transient middle cerebral artery occlusion (tMCAO) were compared with Sham and/or untreated controls groups. The stroke-induced lesion was evaluated by magnetic resonance imaging (MRI). Nicotine (2 mg/kg/12 h) and the AMPK activator AICAR (500 mg/kg/day) were given subcutaneously upon reperfusion until the end of the follow-up period to tMCAO rats. Control sEVs or sEVs loaded with a plasmid encoding a dominant negative isoform of AMPKα2 (AMPKα2-DN) were administered intravenously twice after reperfusion to tMCAO mice. Molecular pathways were analyzed by western blotting. Bederson and open-field tests were applied to evaluate behavioral parameters. RESULTS: Our MRI findings indicated that nicotine treatment reduced brain ischemic injury and improved neurological recovery, as demonstrated by Bederson test, through the inhibition of brain AMPK in ischemic rats. The AMPK activator AICAR reversed the effect of nicotine on injury size and neurological improvement, indicating that the neuroprotective action was dependent on AMPK inhibition. In addition, treatment with AMPKα2-DN sEVs reduced brain lesion and improved neurological recovery. CONCLUSIONS: Our findings demonstrate that the regulation of brain AMPK provides an adequate neuroprotective target for cerebral ischemia, and that the sEV-mediated regulation of this kinase could be a potential clinical strategy against ischemic stroke. Further work, involving scalability in sEV production, immunogenicity, safety and efficacy will be demanding to develop effective and secure therapeutic strategies utilizing sEVs in clinical settings against ischemic stroke.