Hsp31 belongs to the PfpI/Hsp31/DJ-1 superfamily, and has been reported to display chaperone, peptidase and glutathione-independent glyoxalase activities. Here, we show that Hsp31 repairs glyoxal- and methylglyoxal-glycated amino acids and proteins and releases repaired proteins and lactate or glycolate, respectively. Hsp31 deglycates cysteine, arginine and lysine by acting on early glycation intermediates (hemithioacetals and aminocarbinols) and prevents the formation of Schiff bases and advanced glycation endproducts. Hsp31 repairs glycated serum albumin, glyceraldehyde-3-phosphate dehydrogenase, fructose biphosphate aldolase and aspartate aminotransferase. Moreover, we show that bacterial extracts from the hchA mutant display increased glycation levels and that the apparent glyoxalase activity of Hsp31 reflects its deglycase activity. Our results suggest that other Hsp31 members, previously characterized as glutathione-independent glyoxalases, likely function as protein deglycases. (C) 2015 Elsevier Inc. All rights reserved.