A Klebsiella pneumoniae strain, KU6500, which showed resistance to extended-spectrum beta-lactams and produced the plasmid-encoded AmpC beta-lactamase CMY-4, was identified from clinical isolates in Japan. The aim of this study was to identify the mechanism of the high-level expression of blaCMY-4. Sequence analysis indicated that the promoter element of Citrobacter freundii was conserved, but the insertion sequence ISEcp1 coding with the putative promoter element, was inserted into the AmpR binding site. We determined the influence of the promoter on blaCMY-4 expression and beta-lactam resistance. Two recombinant plasmids containing the entire blaCMY-4 gene, with or without the ISEcp1-mediated promoter sequences, were constructed and named pMWampC and pMWISEcp1, respectively. Escherichia coli DH5alpha (pMWISEcp1) was resistant to almost all beta-lactams tested and E. coli DH5alpha (pMWampC) was susceptible to all, except for cephalothin. In addition, the activity of each promoter was measured by subcloning the element into a promoterless luciferase plasmid pGL3-Basic vector. The expression of the putative promoter of ISEcp1 was 18.9-fold higher than that of C. freundii. These results suggest that the putative promoter element of ISEcp1 is necessary for the high-level expression of blaCMY-4 to confer resistance to extended-spectrum cephalosporins.