Acute ischemic stroke, also referred to as a cerebrovascular accident, occurs when localized tissue faces ischemia, leading to an inability to maintain physiological conditions or even resulting in death due to the obstruction or rupture of blood vessels in the brain. The current use of rt-PA for stroke medication has a limited time frame of 3-4.5 hours and is associated with neurotoxicity. Hence, there is an urgent need to explore alternative treatments for ischemic stroke. Cinchonidine, an alkaloid derived from the bark of cinchona, has previously demonstrated anti-inflammatory and antioxidant properties. Notably, cinchonidine exhibits lower toxicity and higher activity compared to other Cinchona alkaloids. Recent studies have highlighted its cell-protective capabilities in endothelial cells stimulated by uremic toxins. This research aims to assess the neuroprotective effects of cinchonidine using a middle cerebral artery occlusion (MCAO)/reperfusion mice model. The findings indicate that pretreatment with cinchonidine (10 mg/kg) significantly reduces brain infarction and improves neurological severity scores, locomotor activity, and rotarod performance in MCAO mice. Moreover, cinchonidine (10 mg/kg) demonstrates a substantial protective effect on blood-brain barrier breakdown following MCAO surgery. In vitro assays reveal that cinchonidine administration rescues cerebral endothelial cells from damage and apoptosis induced by oxygen-glucose deprivation. Furthermore, cinchonidine exhibits anti-neuro-inflammatory properties in the ipsilateral cortex of MCAO mice and in LPS-stimulated microglia. In summary, this study suggests that cinchonidine may mitigate ischemic brain injury through its cellular protective and anti-inflammatory characteristics.