Myocardial ischemia/reperfusion injury (MIRI) refers to heart muscle failure due to cellular and tissue structural damage following coronary revascularization. Myocardial I/R injury is increasing in cats and dogs, kept as pets. The occurrence of MIRI is complicated, and various pathways are possible. Therefore, it is urgent to further explore the mechanism of disease occurrence and development and find key regulatory factors to achieve the goal for reducing MIRI. In this study, MIRI model was established in SD rats through coronary artery ligation and randomized the rats into the model group (MIRI), the trimetazidine group (TMZ), the trimetazidine combined with diazepam treatment group (TMZ+DZ), and the trimetazidine combined with 5-hydroxytryptamine treatment group (TMZ+5-HD). Experimental results indicated that TMZ+DZ significantly reduced myocardial cell apoptosis and inflammation levels in MIRI rats, lowered Creatine Kinase (CK), Creatine Kinase MB Isoenzyme (CK-MB), Lactate Dehydrogenase (LDH), and ROS levels, and decreased myocardial infarct size and mass. While TMZ and TMZ+5-HD also improved these indicators, the therapeutic effect of TMZ+DZ was notably superior to the other two treatment groups. The results suggest that the combination therapy of trimetazidine and diazepam ameliorated myocardial damage in MIRI rats, providing experimental evidence for the treatment of MIRI with the combination therapy of trimetazidine and diazepam.