Mitochondrial glutathione (mGSH) is a critical antioxidant and detoxifying enzyme in mitochondria, playing a central role in maintaining the appropriate redox environment to prevent or repair oxidative modifications that lead to mitochondrial dysfunction and cell death. mGSH is essential for counteracting hydrogen peroxide, lipid hydroperoxides, and xenobiotics, primarily as a cofactor for enzymes like glutathione peroxidase and glutathione-S-transferase (GST). The importance of mGSH is highlighted by its abundance and versatility in mitigating oxidative stress, which is often induced by various pathologies such as hypoxia, ischemia/reperfusion injury, aging, liver diseases, and neurologic disorders. The synthesis of GSH occurs in the cytosol and is regulated by the rate-limiting step of forming γ-glutamylcysteine, which is feedback-inhibited by GSH itself. GSH is distributed in various intracellular organelles, including the endoplasmic reticulum, nucleus, and mitochondria, each maintaining distinct redox pools. In mitochondria, mGSH is primarily found in its reduced form and is crucial for protecting mitochondrial membranes from oxidative damage. mGSH transport into mitochondria is facilitated by carrier-mediated transporters, such as the dicarboxylate and 2-oxoglutarate carriers, which overcome the electrochemical gradient. The concentration of mGSH in mitochondria is similar to that in the cytosol but is regulated by factors like membrane dynamics and cholesterol content. mGSH plays a key role in apoptosis, both extrinsic and intrinsic pathways, by regulating the release of cytochrome c and the activation of caspases. It also controls cardiolipin oxidation, which is essential for mitochondrial membrane stability and cytochrome c binding. In pathological settings, mGSH depletion can sensitize cells to oxidative stress and promote cell death. For example, in hypoxia and reperfusion injury, mGSH depletion can enhance ROS generation and sensitize tumor cells to hypoxia. In liver diseases, mGSH depletion can contribute to steatohepatitis and cirrhosis. In neurologic disorders, mGSH depletion is implicated in Alzheimer's disease and Parkinson's disease, where it affects mitochondrial function and oxidative stress. Strategies to restore or prevent mGSH depletion may have therapeutic significance in treating various pathologies, including cancer, liver diseases, and neurodegenerative disorders.Mitochondrial glutathione (mGSH) is a critical antioxidant and detoxifying enzyme in mitochondria, playing a central role in maintaining the appropriate redox environment to prevent or repair oxidative modifications that lead to mitochondrial dysfunction and cell death. mGSH is essential for counteracting hydrogen peroxide, lipid hydroperoxides, and xenobiotics, primarily as a cofactor for enzymes like glutathione peroxidase and glutathione-S-transferase (GST). The importance of mGSH is highlighted by its abundance and versatility in mitigating oxidative stress, which is often induced by various pathologies such as hypoxia, ischemia/reperfusion injury, aging, liver diseases, and neurologic disorders. The synthesis of GSH occurs in the cytosol and is regulated by the rate-limiting step of forming γ-glutamylcysteine, which is feedback-inhibited by GSH itself. GSH is distributed in various intracellular organelles, including the endoplasmic reticulum, nucleus, and mitochondria, each maintaining distinct redox pools. In mitochondria, mGSH is primarily found in its reduced form and is crucial for protecting mitochondrial membranes from oxidative damage. mGSH transport into mitochondria is facilitated by carrier-mediated transporters, such as the dicarboxylate and 2-oxoglutarate carriers, which overcome the electrochemical gradient. The concentration of mGSH in mitochondria is similar to that in the cytosol but is regulated by factors like membrane dynamics and cholesterol content. mGSH plays a key role in apoptosis, both extrinsic and intrinsic pathways, by regulating the release of cytochrome c and the activation of caspases. It also controls cardiolipin oxidation, which is essential for mitochondrial membrane stability and cytochrome c binding. In pathological settings, mGSH depletion can sensitize cells to oxidative stress and promote cell death. For example, in hypoxia and reperfusion injury, mGSH depletion can enhance ROS generation and sensitize tumor cells to hypoxia. In liver diseases, mGSH depletion can contribute to steatohepatitis and cirrhosis. In neurologic disorders, mGSH depletion is implicated in Alzheimer's disease and Parkinson's disease, where it affects mitochondrial function and oxidative stress. Strategies to restore or prevent mGSH depletion may have therapeutic significance in treating various pathologies, including cancer, liver diseases, and neurodegenerative disorders.