Mitochondria are the primary intracellular site of oxygen consumption and the major source of reactive oxygen species (ROS), most of them originating from the mitochondrial respiratory chain. Among the arsenal of antioxidants and detoxifying enzymes existing in mitochondria, mitochondrial glutathione (mGSH) emerges as the main line of defense for the maintenance of the appropriate mitochondrial redox environment to avoid or repair oxidative modifications leading to mitochondrial dysfunction and cell death. mGSH importance is based not only on its abundance, but also on its versatility to counteract hydrogen peroxide, lipid hydroperoxides, or xenobiotics, mainly as a cofactor of enzymes such as glutathione peroxidase or glutathione-S-transferase (GST). Many death-inducing stimuli interact with mitochondria, causing oxidative stress; in addition, numerous pathologies are characterized by a consistent decrease in mGSH levels, which may sensitize to additional insults. From the evaluation of mGSH influence on different pathologic settings such as hypoxia, ischemia/reperfusion injury, aging, liver diseases, and neurologic disorders, it is becoming evident that it has an important role in the pathophysiology and biomedical strategies aimed to boost mGSH levels.