〔Abstract〕 To explore the effects of transmembrane protein 16B(TMEM16B) on brain edema and blood-brain barrier after cerebral ischemia-reperfusion injury in rats. Methods TMEM16B overexpression and knockdown was performed by adeno-associated virus (AAV), and then adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO). Rats were randomly divided into Sham group, MCAO group, AAV no-load group, TMEM16B overexpression group and TMEM16B knockdown group. Modified neurological severity scores, adhesive removal test and cylinder test were used to evaluate neurologic function.The ultrastructure of ischemic brain tissue was observed by transmission electron microscope. Brain water content was reflected by dry wet weight ratio of brain tissue. The expressions ofTMEM16B, aquaporin4(AQP4), Claudin5 and zonula occludens-1 (ZO-1) were investigated by immunofluorescence and Western blot. Results Compared with the AAV no-load group, the sensory and motor functions of rats in TMEM16B overexpression group were significantly impaired. Mitochondria were swollen; mitochondrial cristae and tight junctions disappeared. The brain water content was higher in overexpression group. The expression ofTMEM16B and AQP4 increased while the expression of Claudin5 and ZO-1 decreased. Compared with the AAV no-load group, the rats in TMEM16B knockdown group showed some recovery in motor function. The mitochondrial cristae and structure were clear, and the basement membrane was partially blurred. The brain water content was lower in knockdown group. The protein levels ofTMEM16B and AQP4 were lower while the levels ofClaudin5 and ZO-1 were higher than that in AAV no-load group. Conclusion An increase in TMEM16B aggravates brain edema and blood-brain barrier damage in rats after cerebral ischemia-reperfusion injury, while a decrease in TMEM16B expression alleviates brain edema and protects the blood-brain barrier.