Gram-negative bacteria producing VIM-type metallo-β-lactamases (MBLs) are increasingly isolated worldwide. The VIM group includes at least 13 variants, and blaVIM-12 is a recently identified blaVIM-1/VIM-2 hybrid gene originally found in 2005 in a Klebsiella pneumoniae clinical isolate from Greece (residing in class 1 integron In-h12). It had not been detected in other gram-negative species, and the kinetic parameters of the purified VIM-12 enzyme were uncharacterized. We identified the blaVIM-12 gene in an Escherichia coli clinical isolate (strain 28) recovered in February 2006 from a decubitus ulcer infection of an 85-year-old male hospitalized in Thessaloniki, Greece. Strain 28 was susceptible to imipenem, meropenem, aztreonam, ciprofloxacin, and cotrimoxazole but resistant to other antimicrobials. MBL production was confirmed by positive imipenem-EDTA double disc synergy test (DDST) and Etest MBL. PCR and sequencing showed strain 28 carried blaVIM and blaCMY/LAT genes (negative for other β-lactamase genes) and an integron structure identical to In-h12. Filter mating experiments yielded transconjugants with elevated MICs of penicillins, cephalosporins, and aminoglycosides, harboring a ~70 kb plasmid positive for blaCMY/LAT (not blaVIM). Restriction fragment length polymorphism (RFLP) showed the plasmid of strain 28 differed from p2873 (carrying blaVIM-12 in K. pneumoniae). Southern blotting revealed blaVIM-12 was chromosomally located. This is the first documentation of VIM-12 (a VIM-1/VIM-2 hybrid MBL) production in E. coli. Several blaVIM-12-producing K. pneumoniae strains were identified in the same hospital since 2005, indicating wide dissemination. E. coli strain 28 carried In-h12 on the chromosome (not on transferable plasmid p2873). We speculate In-h12 arose in the hospital setting (where blaVIM-1- and blaVIM-2-carrying pathogens are common) or blaVIM-12 has a wide natural distribution.