A carbapenem-resistant Serratia marcescens strain, 10mdr148, was identified in a Japanese hospital in 2010. The carbapenem resistance of this strain was attributed to the production of a novel metallo-β-lactamase (MBL), named SMB-1 (Serratia metallo-β-lactamase). SMB-1 possessed a zinc binding motif, H(Q)XHXDH (residues 116 to 121), H196, and H263 and was categorized as a member of subclass B3 MBL. SMB-1 has 75% amino acid identity with the most closely related MBL, AMO1, of uncultured bacterium, recently identified through the metagenomic analysis of apple orchard soil. The introduction of bla(SMB-1) into Escherichia coli conferred resistance to a variety of β-lactam antibiotics, penicillins, cephalosporins, and carbapenems, but not aztreonam, a resistance pattern consistent with those of other MBLs. SMB-1 demonstrated high k(cat) values of >500 s(-1) for carbapenems, resulting in the highest hydrolyzing efficiency (k(cat)/K(m)) among the agents tested. The hydrolyzing activity of SMB-1 was well inhibited by chelating agents. The bla(SMB-1) gene was located on the chromosome of S. marcescens strain 10mdr148 and at the 3' end of the ISCR1 element in complex with a typical class 1 integron carrying aac(6')-Ib and catB3 gene cassettes. Downstream of bla(SMB-1), the second copy of the 3'conserved segment and ISCR1 were found. To our knowledge, this is the first subclass B3 MBL gene associated with an ISCR1 element identified in an Enterobacteriaceae clinical isolate. A variety of antibiotic resistance genes embedded with ISCR1 have been widely spread among Enterobacteriaceae clinical isolates, thus the further dissemination of bla(SMB-1) mediated by ISCR1 transposition activity may become a future concern.