Objective To evaluate influences of fentanyl and sufentanil on the cardioprotection effect of preemptive levothyroxine-sodium administration in the rats. Methods immature rats were randomly allocated into seven groups (n=8): blank control group (group BC), control group (group C), levothyroxine-sodium 10μg group (group 10μg), fentanyl group (group F), sufentanil group (group S), combined fentanyl and levothyroxine-sodium group (group F+L) and combined sufentanil and levothyroxine-sodium group (group S+L). The isolated heart model was established with Langendorff apparatus. Hearts in the groups F and F+L were perfused with KH fluid containing fentanyl 30μg/L, and hearts in the groups F and F+L were perfused with KH fluid containing sufentanil 3μg/L. Results The hemodynamic variables at 30 min of the reperfusion were inferior in the groups F and S than in the group BC, but better in the groups 10μg, F+L and S+L than in the groups C, F, and S. The coronary perfusion flows in the groups BC, C, 10μg, F, S, F+L and S+L were 19±2, 16±1, 21±2, 17±1, 17±1, 21±1, 22±1 ml/min, respectively, with a significantly higher coronary perfusion flow in the groups 10μg, F+L and S+L compared to the groups C, F and S. The myocardial bound creatine kinase level in the coronary perfusion fluid during the reperfusion period was significantly higher in the groups C, F and S compared with groups 10μg, F+L and S+L. The expression of both HSP70 proteins and MHC mRNA in the myocardium was significantly stronger in the groups 10μg, F+L and S+L than in the groups C, F and S. However, there were no differences in all the observed parameters among groups 10μg, F+L and S+L. Conclusion Both fentanyl and sufentanil do not produce significantly influence on the cardioprotective effect of levothyroxine sodium preconditioning.