Reactive oxygen species (ROS) are not only damaging molecules but are also involved in redox signaling events. In mitochondria, thiol redox state is finely tuned mainly by the thioredoxin and the glutathione systems. Cyclophilin D (CypD), a key regulator of mitochondrial permeability transition, was recently shown to be subjected to redox regulation. In particular, CypD was recognized to directly interact with thioredoxin 2 and peroxiredoxin 3 of the thioredoxin system. In addition, the CypD prolyl cis-trans isomerase activity was shown to depend on its redox state with a higher catalytic efficiency in its reduced status. An important player in mitochondrial thiol redox regulation is glutaredoxin 2 (Grx2). This enzyme normally resides in the mitochondrial matrix as an inactive dimer coordinating a 2Fe-2S2+ cluster. However, oxidative stress induction can trigger protein monomerization and activation. In this context, it has been observed that Grx2 is extremely active as a disulfide reductase in conditions of glutathione depletion and thioredoxin system inhibition. Thus, Grx2 seems very efficient in the recovery of a proper redox balance. In conclusion, thiol redox regulation, by tuning the activity of specific enzymes, has a key role in mitochondrial signaling pathways.