Transglutaminase 2 (TGase2) is a ubiquitously expressed enzyme that catalyzes irreversible post-translational modification of protein, forming cross-linked protein aggregates. We previously reported that intracellular TGase2 is activated by oxidative stress. To elucidate the functional role of TGase2 activation in cells under the oxidatively stressed condition, we identified the mediator that activates TGase2. In this study, we showed that low levels of oxidative stress trigger the release of TGF, which subsequently activates TGase2 through the nuclear translocation of Smad3. Analysis of substrate proteins reveals that TGase2-mediated protein modification results in a decrease of protein solubility and a collapse of intermediate filament network, which leads to aggregation of proteins. We confirm these results using lens tissues from TGase2-deficient mice. Among several antioxidants tried, only N-acetylcysteine effectively inhibits TGF-mediated activation of TGase2. These results indicate that TGF mediates oxidative stress-induced protein aggregation through activation of TGase2 and suggest that the formation of protein aggregation may not be a passive process of self-assembly of oxidatively damaged proteins but may be an active cellular response to oxidative stress. Therefore, TGF-TGase2 pathway may have implications for both the pathogenesis of age-related degenerative diseases and the development of pharmaceutics.—Shin, D.-M., Jeon, J.-H. Kim, C.-W., Cho, S.-Y., Lee, H.-J., Jang, G.-Y., Jeong, E. M., Lee, D.-S., Kang, J.-H., Melino, G., Park, S.-C., Kim, I.-G. TGF mediates activation of transglutaminase 2 in response to oxidative stress that leads to protein aggregation.