To investigate that penehyclidine hydrochloride (PHC) plays a cardiac protection role in myocardial ischemia/reperfusion injury (IRI) through mitochondrial dynamics mechanism. Rat model of myocardial I/R injury was established by the ligation of left anterior descending coronary artery for 30 min followed by 3 h perfusion. Before I/R, the rats were pretreated with or without PHC. Cardiac function was measured by echocardiography. The activities/levels of myocardial enzymes, oxidants and antioxidant enzymes were detected. Evans blue/TTC double staining was performed to assess infarct size. Cell apoptosis was evaluated by TUNEL assay. Western blot and real time fluorescent quantitative PCR was performed to analyze the expression of Drp(1), Mfn(1), Mfn(2). Meanwhile, the rats were given a single injection of PHC before I/R. The effects of PHC on myocardial infarct and cardiac function were investigated after 7 days post-reperfusion. Our results showed that PHC pretreatment improved imbalance of mitochondrial dynamics induced by oxidative stressor in IRI. PHC preconditioning alleviated apoptotic rate of cell by improving the imbalance of mitochondrial dynamics in IRI. Meanwhile, we showed that PHC remarkably improved cardiac function, myocardial injury by decreasing infarct size and attenuated levels of myocardial enzyme. Additionally, PHC also exerted long-term cardiac protection in a rat model of I/R injury by decreasing infarct size and improving cardiac function. These results suggested that PHC could efficiently protect the rats against I/R-induced myocardial injury via the mechanism of mitochondrial dynamics.