Isothiocyanates (ITCs) are produced by cruciferous plants to protect them against herbivores and infection by microbes. These compounds are of particular interest due to their antimicrobial and anticarcinogenic properties. The breakdown of ITCs in nature is catalyzed by isothiocyanate hydrolase (ITCase)， a novel family within the metallo-β-lactamase-fold superfamily of proteins. genes that code for ITCases are particularly widespread in insect- and plant-associated bacteria. Enzymatic characterization of seven phylogenetically related but distinct ITCases exhibited similar activities on six selected ITCs， suggesting that phylogenetic diversity does not determine the substrate specificity of ITCases. X-ray crystallography studies of two ITCases sharing 42% amino acid sequence identity revealed a highly conserved tertiary structure. Notable features of ITCase include a hydrophobic active site with two Zn ions coordinating water/hydroxide， and a flexible cap that is implicated in substrate recognition that covers the active site. This report reveals the function and structure of the previously uncharacterized family of isothiocyanate hydrolases within the otherwise relatively well-studied superfamily of metallo-β-lactamases.: This study explores a newly discovered protein in the beta-lactamase superfamily: SaxA， or isothiocyanate hydrolase. Isothiocyanates are defensive compounds found in many cabbage-related crop plants， and are currently being investigated for their antimicrobial and anticarcinogenic properties. We show that isothiocyanate hydrolases are responsible for the breakdown of several of these plant defensive chemicals and suggest their potential for mitigating the beneficial effects of isothiocyanates in crop protection and cancer prevention.