In response to cellular redox balance, we show that the 20S proteasome from yeast (Saccharomyces cerevisiae) is glutathionylated in vivo. We found a positive correlation among proteasomal glutathionylation, protein carbonyl generation and loss of intracellular reductive capability, demonstrated by the GSH/GSSG rate. Purified proteasome from cells grown to stationary phase into glucose-containing medium exhibited higher levels of glutathionylation when compared to the proteasome extracted from glycerol-containing medium. The oxidation of cysteinyl residues (Cys-SH) to sulfenic acid (Cys-SOH) turns them susceptible to be modified by S-glutathionylation (Cys-S-SG), decreasing the proteasome chymotrypsin-like activity, although no effect upon trypsin-like activity was observed when compared to the reduced protein. However, the glutathionylated proteasome showed higher proteolysis levels against either oxidized or naturally unfolded proteins as substrates. Our studies indicate that the chymotrypsin-like activity was not the most important activity against those proteins whose breakdown counts on a strong cooperation of the others activities. These data suggest that protesome glutathionylation seems to be involved not only in the protection of cysteinyl hiperoxidation, but it also plays a role as a regulatory pathway through its enzymatic activities during oxidative stress by degrading oxidized proteins and allowing cells to reestablish the metabolism. As the redox balance is normalized, the proteasome is enzimatically reduced by proteins such as glutaredoxins and thioredoxins, restoring its basal activity.