The mitochondrial respiratory chain represents the major source of reactive oxygen species (ROS) in cells and its dysfunction may contribute to the pathogenesis of several diseases. In mitochondria, glutathione is the major redox buffer and is a good indicator for the redox environment of the cell. Indeed, overproduction of ROS decreases the ratio between reduced and oxidized glutathione leading the latter to bind to proteins by a mechanism called glutathionylation. In this study, we demonstrate that in isolated cardiac mitochondria the respiratory chain enzyme Complex I is highly susceptible to glutathionylation under conditions of oxidative stress, showing a significant dose- and time-dependent decrease of the activity after treatment with oxidized glutathione. Among respiratory chain enzymes, Complex I appears the most affected by the oxidant-mediated inactivation in isolated mitochondria. Also, in cultured cardiomyocytes CI activity was strongly inhibited after in vivo treatment with hydrogen peroxide. Noteworthy, HPLC analysis showed a significant increase of protein glutathionylation in oxidatively stressed cells and this rise is in vivo reverted after incubation of cells with anti-oxidant N-acetyl-cysteine. These findings take particular importance given that CI represents the entry point of electrons into oxidative phosphorylation and that the threshold at which CI dysfunction affects ATP production is lower than that of any other OXPHOS complexes, making the enzyme particularly critical for the health of cells.