Abstract: AIM　To study the effects of butylphthalide(NBP) on the function and ultrastructure of neuronal mitochondria during cerebral ischemia. METHODS　Cerebral ischemia models of rat middle cerebral artery occlusion in vivo and primarily cultured neurons subjected to hypoxia/hypoglycemia in vitro were used. The mitochondria was prepared for measurement of mitochondria membrane fluidity(MMF), mitochondria membrane potential(MMP) and the total activity of mitochondria ATPase. MMF was assessed with fluorescent probes diphenylhexatriene(DPH) and MMP was measured with the fluorescence of loaded rhodamine-123 using flow cytometry. Electron microscopy was also used to study the morphological changes of neuronal mitochondria. RESULTS　The value of η was enhanced significantly in the vehicle(MCAO) group. It means that the MMF was decreased deeply during the early stage of cerebral ischemia. The decrease of MMP and total ATPase activity were found in rat fetal neurons subjected to 3 h-hypoxia/hypoglycemia. The results indicate that after pretreatment with dl-NBP(5 mg.kg^-1 and 10 mg.kg^-1 ip), the MMF was close to that of the control level. The decrease of MMP and ATPase activity were not found following treatment with dl-, l-, and d-NBP(10 μmol.L^-1). In addition, NBP was found to improve the severe swelling and marked vacuolation of mitochondria in morphology. CONCLUSION　The results suggest that the improving effects of NBP on mitochondrial injury and morphological changes might contribute to its therapeutic action on experimental stroke.