Aim To study the role of nitric oxide (NO) in the preconditioning of the heart with hyperthemia in rats. Methods The isolated rat heart was perfused in a Langendorff model. Hearts for all groups were subjected to 4 h hypothermia (4℃) and 40 min reperfusion. Heat stress was induced by whole-body hyperthermia (rectal 42℃, 15 min) 24 h before the experiment. Heart rate, coronary flow, left ventricular pressure, and its derivative (±dp/dt max ) were recorded, and tumor necrosis factor-alpha (TNF-α) immunoreactivity in myocardial tissues and the activity of creatine kinase (CK) in the coronary effluent were measured. Results Pretreatment with hyperthermia significantly improved the recovery of cardiac protection, reduced the release of CK, and increased plasma concentrations of NO. Pretreatment with L-NAME, an inhibitor of NOS, abolished the protective effects and the increased level of TNF-α and NO elicited by hyperthermia. Conclusion Endogenous NO is involved in the cardioprotection afforded by heat stress, and the beneficial effects of NO are related with stimulation of TNF-α production in the rat heart.