Objective To study the effects of Rubia yunnanensis on NO, oxidative stress and mitochondrial energy metabolism in myocardial ischemia/reperfusion injury. Methods 48 male Wistar rats were randomly divided into sham operation group, model group, Fufang Danshen Tablet group, low, middle and high doses of Rubia yunnanensis groups. After administrated for 14 d, all rats except the sham group were made myocardial ischemia/reperfusion injury model by ligating the left anterior descending branch of the coronary artery for 30 min and reperfusion for 2 h. The content of NO and the activity of eNOS in serum, and the levels of ROS, CAT, T-AOC and ATP in myocardial tissue, and the changes of mitochondrial ATPase and membrane potential in myocardial cells were detected. Results Compared with the sham group, the contents of NO, CAT, T-AOC, the activity of eNOS, the mitochondrial energy metabolizing ATPase and the mitochondrial membrane potential of cardiomyocytes in the model group decreased, and the level of ROS in myocardial tissue increased (P<0.05). There were no significant difference in Na+-K+-ATPase and mitochondrial membrane potential both in low Rubia yunnanensis dose group and model group, while the content of NO, the activities of CAT, T-AOC, eNOS, mitochondrial energy metabolizing ATPase and mitochondrial membrane potential in Fufang Danshen Tablet group and other Rubia yunnanensis treatment groups were all increased, and the level of ROS were decreased (P<0.05). Conclusion Rubia yunnanensis has protective effect on myocardial ischemia/reperfusion injury, and its mechanism may be related to the increase of serum NO level, antioxidation and improvement of mitochondrial energy metabolism.