Metallo-β-lactamases (MBLs) hydrolyze most β-lactam antibiotics, and bacteria containing this kind of enzyme pose a serious threat to the public health. The newly identified New Delhi MBL (NDM-1) is a new member of this family that shows tight binding to penicillin and cephalosporins. The rapid dissemination of NDM-1 in clinically relevant bacteria has become a global concern. However, no clinically useful inhibitors against MBLs exist, partly due to the lack of knowledge about the catalysis mechanism of this kind of enzyme. Here we report the crystal structure of this novel enzyme in complex with a hydrolyzed ampicillin at its active site at 1.3-Å resolution. Structural comparison with other MBLs revealed a new hydrolysis mechanism applicable to all three subclasses of MBLs, which might help the design of mechanism based inhibitors.