Abstract The mitochondrial pool of GSH (glutathione) is considered the major redox system in maintaining matrix redox homeostasis, preserving sulfhydryl groups of mitochondrial proteins in appropriate redox state, protecting mitochondrial DNA against mitochondrial-derived ROS, and in defending mitochondrial membranes against oxidative damage. Despite its importance in maintaining mitochondrial functionality, GSH is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. In this work we found that SLG (S-D-lactoylglutathione), an intermediate of the glyoxalase system, can enter the mitochondria and there be hydrolyzed from mitochondrial glyoxalase II enzyme to D-lactate and GSH. We demonstrated SLG transport from cytosol to mitochondria by incubating substrates with radioactive compounds that showed two different kinetic curves for SLG or GSH substrates, indicating different kinetic transport. The incubation of functionally and intact mitochondria with SLG showed increased GSH levels in normal mitochondria and in artificially uncoupled mitochondria, demonstrating transport not linked to ATP presence. Also mitochondrial-swelling assay confirmed SLG entrance into organelles. Moreover we observed oxygen uptake and generation of membrane potential probably linked to D-lactate oxidation which is a product of SLG hydrolysis. The latter data were confirmed by oxidation of D-lactate in mitochondria evaluated by measuring mitochondrial D-lactate dehydrogenize activity. In this work we also showed the presence of mitochondrial glyoxalase II, the enzyme that catalyzes SLG hydrolysis, in intermembrane space and mitochondrial matrix. In conclusion, this work showed new alternative sources of GSH supply to the mitochondria by SLG, an intermediate of the glyoxalase system.