Herba Cistanche, a Chinese herb derived from the whole plant of Cistanche deserticola Y.C. Ma (Orobanchaceae), has been shown to enhance mitochondrial ATP generation and to protect against myocardial ischemia/reperfusion (I/R) injury ex vivo in rats. To define the role of mitochondria in the cardioprotective action of Herba Cistanche, we investigated the effect of Herba Cistanche treatment on mitochondrial glutathione status and functional parameters in rat hearts. Treatment with a methanol extract of Herba Cistanche enhanced mitochondrial glutathione status, decreased mitochondrial Ca(2+) content, and increased mitochondrial membrane potential. In addition, an increase in State 4 respiration, indicative of uncoupled respiration, was observed in mitochondria isolated from Herba Cistanche-treated rat hearts. The enhancement of mitochondrial glutathione status and functional ability, as well as the putative induction of uncoupling proteins, may be related to cardioprotection afforded by Herba Cistanche treatment protecting against I/R injury.