Significance The genomes of the vast majority of eukaryotes encode a protein [named nitrilase-like protein 1 (Nit1) in humans and mice] whose enzymatic function has long been unknown. We show here that the mammalian Nit1 and the corresponding yeast protein efficiently hydrolyze the deaminated form of the common intracellular antioxidant glutathione. In turn, deaminated glutathione can be produced by a side activity of numerous transaminases. Thus, Nit1 repairs an undesired product, arising from the slow (and erroneous) transformation of an important metabolite by some abundant intracellular enzymes. The importance of this Nit1 function is underscored by the finding that enzymes with the same activity occur in Escherichia coli and in other glutathione-producing bacteria.