Liquiritigenin (Lq) offers cytoprotective effects against various cardiac injuries, but its beneficial effects on myocardial ischemic (MI) injury and the related mechanisms remain unclear. In the in vivo study, an animal model of MI was induced by intraperitoneal injection of isoproterenol (Iso, 85 mg/kg). ECG, heart rate, serum levels of CK and CK-MB, histopathological changes, and reactive oxygen species (ROS) levels were all measured. In vitro, H9c2 cells were divided into four groups and treated for 24 hr with liquiritigenin (30 μmol/L and 100 μmol/L) followed with CoCl2 (800 μmol/L) for another 24 hr. Cell viability, apoptosis, mitochondrial membrane potential, and intracellular Ca2+ concentration ([Ca2+ ]i ) were then assessed. The L-type Ca2+ current (ICa-L ) was detected using a patch clamp technique on isolated rat ventricular myocytes. The myocyte contraction and Ca2+ transients were measured using an IonOptix detection system. The remarkable cardiac injury and generation of intracellular ROS induced by Iso were alleviated via treatment with Lq. CoCl2 administration induced cell apoptosis, mitochondrial dysfunction, and Ca2+ overload in H9c2; Lq reduces these deleterious effects of CoCl2 . Meanwhile, Lq blocked ICa-L in a dose-dependent manner. The half-maximal inhibitory concentration of Lq was 110.87 μmol/L. Lq reversibly reduced the amplitude of cell contraction as well as the Ca2+ transients. The results show that Lq protects against MI injury by antioxidation, antiapoptosis, counteraction mitochondrial dysfunction, and inhibition of ICa-L , thus damping intracellular Ca2+ .© 2022 John Wiley & Sons Ltd.