Ethnopharmacological relevance: Tongxinluo (TXL) is a multifunctional traditional Chinese medicine and has been widely used in the treatment of cardiovascular and cerebrovascular diseases. Numerous studies demonstrate that TXL is a novel neuroprotective drug, however, the mechanisms are largely unknown. Aim of the study: we aimed to demonstrate the protective effect of TXL on cerebral ischemia/reperfusion (I/R) injury and provide the evidence for the involvement of Connexin 43/Calpain II/ Bax/Caspase-3 pathway in TXL-mediated neuroprotection. Methods: Focal cerebral I/R injury were induced by transient middle cerebral artery occlusion (MCAO, for 90 min) in adult male Sprague-Dawley rats. We estimated the effects of TXL on I/R injury including neurological deficit assessment and cerebral infarct volume measurement via TTC staining, and detected the protein expression of Connexin 43 (Cx43) by western blot. Furthermore, after the intracerebroventricular injection of carbenoxolone (CBX, the inhibitor of Cx43) at 30 min before MCAO surgery, Calpain II, Bax and cleaved Caspased-3 immunoreactivity in ischemic penumbra region was detected by immunofluorescent staining, and cell apoptosis was detected by TUNEL staining. Results: TXL treatment greatly improved neurological deficit and reduced the infarction volume compared to MCAO with buffer treatment (P < 0.05), and TXL pre-post treatment showed better results than TXL pretreatment. TXL pre-post treatment significantly up-regulated Cx43 protein expression at 3d, 7d and 14d post injury compared to MCAO with buffer treatment (P < 0.05). Meanwhile, the immunoreactivity of Calpain II, Bax and cleaved Caspase-3 in ischemic penumbra region was obviously decreased by TXL pre-post treatment compared to MCAO group (P < 0.05). However, with the treatment of the Cx43 inhibitor, CBX, the down regulated effect of TXL on Calpain II, Bax and cleaved Caspase-3 immunoreactivity was abolished (P < 0.05). Moreover, the protective effect of TXL against neuron apoptosis in penumbra region was conteracted by CBX (P < 0.05). Conclusions: TXL could effectively protect against I/R injury and reduced cell death via Cx43/Calpain II/Bax/ Caspase-3 pathway, which contribute to I/R injury prevention and therapy.