The development of effective post-stroke therapy is highly demanded. Medicarpin is a key active component of a famous Chinese herbal prescription used for post-stroke treatment in Taiwan; however, little is known about its biological effects and mechanisms of action. Herein, we implemented a murine model of cerebral ischemic/reperfusional injury-related stroke to elucidate medicarpin's neuroprotective effect. In male ICR mice 24 h after stroke induction, treatment with medicarpin (0.5 and 1.0 mg/kg, i.v.) markedly enhanced the survival rates, improved moving distance and walking area coverage, reduced brain infarction, and preserved the blood-brain barrier, supporting medicarpin's protective effect on stroke-induced injury. Immunohistochemistry analysis further revealed that medicarpin treatment decreased the expression/activation of p65NF-κB and caspase 3, especially near the infarct cortex, while promoting the expression of neurogenesis-associated proteins, including doublecortin (DCX), brain-derived neurotrophic factor (BDNF), and tyrosine receptor kinase B (TrkB). These changes of expression levels were accompanied by GSK-3 inactivation and β-catenin upregulation. Notably, pretreatment with LY294002, a PI3K inhibitor, abolished the aforementioned beneficial effects of medicarpin, illustrating an essential role of PI3K/Akt activation in medicarpin's neuroprotective and reparative activities. In vitro studies revealed that medicarpin displayed strong anti-inflammatory activity by reducing nitric oxide (NO) production in lipopolysaccharide-stimulated microglial cells (BV2) with an IC50 around 5 ±1 (μM) and anti-apoptotic activity in neuronal cells (N2A) subjected to oxygen-glucose deprivation with an IC50 around 13 ± 2 (μM). Collectively, this is the first report to demonstrate that medicarpin, isolated from Radix Hedysari, ameliorates ischemic brain injury through its anti-inflammatory microglia/NO), anti-apoptotic (neuronal cells/OGD) and neuroprotective effects by activating the PI3K/Akt-dependent GSK-3 inactivation for upregulating β-catenin, which in turn decreases the expression/activation of p65NF-κB and caspase 3 and promotes the expression of neurogenic (DCX, BDNF, TrkB) and neuroprotective (Bcl2) factors in the brain.