To probe the effect of reduced form coenzyme Ⅰ(NADH) in antagonizing cardiac muscle toxicity induced by doxorubicin and its underlying mechanism. Primary culture of myocardial cells of SD rat and doxorubicin injury model were established. MTT assay, laser confocal microscopy, transmission electron microscopy and biological oxygen monitor were used to observe the morphology and function of mitochondria. The results showed that the killing rate was increased in the group of doxorubicin, and that in the group of NADH/doxorubicin was greatly decreased. Doxorubicin could induce ultrastructural damage of cardiomyocyte mitochondria, manifested as swelling, disintegration, disruption of cristae and fusion. Cardiac mitochondria were protected against injuries in the group treated with NADH/doxorubicin. There was a significant difference in the fluorescence intensity of mitochondria membrane potentional and ROS between the groups treated with doxorubicin and NADH/ doxorubicin. It is suggested that NADH can significantly antagonizing cardiac muscle toxicity induced by doxorubicin and can protect mitochondrial structure and function.