Though mild hypothermia displays an optimistic alleviation of contractive failure in the ischemia/reperfusion myocardium, we still lacked answers to many questions about its potential mechanisms. Our hypothesis is that hypothermia (32°C) induced in ischemia can ease mitochondrial injury resulting in improvement of myocardial contractility even under the condition of a normothermic reperfusion. Fifty newly born 1-2 d Sprague-Dawley rats were executed and the primary cardiomyocytes were obtained and cultivated in vitro. Myocytes were randomized into three groups and then subjected to ischemia either at 32°C or 37°C, both prior to undergoing reperfusion at 37°C. Contractility was presented as frequency and velocity. Ultrastructural alterations of cardiomyocytes and mitochondrion underwent semi-quantitative analysis with transmission electron microscopy and respiratory function of mitochondria was further assessed simultaneously. During cooling ischemia and following reperfusion, cardiomyocytes acquired a more immediate restoration to baseline level and had a significant difference as compared with those in normothermia (P < 0.05). Furthermore, hypothermia preserved the ultrastructure of myocytes and mitochondrion after ischemia. However, measurement on Heart Injury Score and form factor revealed no differences after 2-h reperfusion either in hypothermia or normothermia. On the contrary, the surface area and respiratory function of mitochondrion in reperfusion differed significantly in both groups (P < 0.05) which had an accordance with the variation on contractile performance. Hypothermia only induced in ischemia can bring contractility benefit even under a normothermia reperfusion in cultured cardiomyocytes.