OBJECTIVE To investigate the protective effects and potential mechanism of penehyclidine hydrochloride （PHC） on myocardial ischemia-reperfusion （I/R） injury in mice through the macrophage migration inhibitory factor （MIF）/adenosine monophosphate-activated protein kinase （AMPK） signaling pathways. METHODS Male C57BL/6 mice were randomly divided into sham operation group， I/R group， I/R+PHC group （PHC 20 μg/kg）， I/R+ISO-1 group （MIF inhibitor 35 mg/kg）， I/R+ PHC+ISO-1 group （with the same dosage as each single drug group）， with 8 mice in each group. Except for the sham operation group， the myocardial I/R injury model was prepared by coronary artery ligation. Thirty minutes before ligation， mice in each drug group were injected with 1 mL of the corresponding drug solution through the tail vein. After 120 min of reperfusion， the levels of cardiac function indexes [heart rate， stroke volume， ejection fraction， cardiac output， left ventricular posterior wall thickness in systole （LVPWs）， left ventricular posterior wall thickness in diastole （LVPWd）]， serum inflammatory factors [interleukin-6 （IL- 6）， IL-10， tumor necrosis factor-α （TNF-α）] in mice were detected in each group； the pathological changes and ultrastructure of myocardial tissue were observed， and the protein expressions of B cell lymphoma-2 （Bcl-2）， phosphorylated AMPKα （p-AMPKα） and MIF in myocardial tissue were detected. RESULTS Compared with the sham operation group， the myocardial cells in the I/R group were loosely arranged， with severe infiltration of inflammatory cells and obvious mitochondrial damage. Serum levels of IL-6 and TNF-α and protein expression of p-AMPKα in myocardial tissue were significantly increased or upregulated， while heart rate， stroke volume， ejection fraction， cardiac output， LVPWd and serum level of IL-10 were significantly decreased （P＜0.05）. Compared with the I/R group， the myocardial tissue lesions in the I/R+PHC group were alleviated； serum levels of IL-6 and TNF-α were decreased significantly， while heart rate， stroke volume， ejection fraction， cardiac output， LVPWs， LVPWd， serum level of IL-10， and protein expressions of Bcl-2， p- AMPKα and MIF in myocardial tissue were significantly increased or upregulated （P＜0.05）. However， myocardial tissue lesions of mice in the I/R+ISO-1 group worsened， with most quantitative indicators significantly deteriorating （P＜0.05）； MIF inhibitor could generally reverse the protective effect of PHC on I/R mice （P＜0.05）. CONCLUSIONS PHC can improve cardiac function， reduce myocardial inflammation， and restore the ultrastructure of myocardial tissue in I/R mice. These effects may be related to the activation of the MIF/AMPK signaling pathway.