||Reperfusion of the previously ischemic myocardium caused a decline in ATP production, mitochondrial Ca2+ overload, reactive oxygen species (ROS) production and mitochondrial permeability transition (PT), all of which can lead to necrosis- and/or apoptosis-mediated cell death. As mitochondria play a crucial role in determining the fate of cardiomyocytes following ischemia/reperfusion (I-R), protection strategy against I-R injury should therefore be targeted on the mitochondria by preserving the mitochondrial structural and functional integrity. Herba Cistanche is a “Yang-invigorating” Chinese tonifying herb that can up-regulate cellular activities by stimulating mitochondrial ATP generation from oxidative phosphorylation. The effects of pretreatment with various Herba Cistanche extracts (methanolic, aqueous and total glycosides fraction) were investigated using an ex vivo rat model of myocardial I-R injury and an in vitro model of menadione-induced toxicity in H9c2 cells. Based on the comparative studies, it is clear that glycosides did not seem to be the active ingredient(s) in producing the ATP-enhancing and cardioprotective effect. For the methanolic and aqueous extract of Herba Cistanche, both of them showed ATP-enhancement and cardioprotection against I-R injury, with the effect of the methanolic extract being more potent. Further investigation showed that these two extracts can also enhance glutathione status, decrease the mitochondrial Ca2+ level as well as increase the mitochondrial membrane potential. The biochemical mechanism involved in the cardioprotective action of Herba Cistanche may be attributed to the preservation of mitochondrial functional integrity during ischemia.