Spacer sequences of 42 and 127 bp, respectively, between the ISEcp1 insertion sequence and blaCTX-M have been observed in Klebsiella pneumoniae. However, it is not known whether different spacers upstream of blaCTX-M and a promoter within the 127 bp spacer influence cephalosporin resistance. Three recombinant plasmids with different spacers and with or without ISEcp1 were constructed to compare blaCTX-M-3 expression and susceptibility to cephalosporins. Our experiment revealed enhanced blaCTX-M-3 expression and a relatively high level resistance to cefotaxime and cefepime in recombinant plasmid IS42CTX-M-3, which contained ISEcp1 and the 42 bp spacer. A minor difference in blaCTX-M-3 expression was observed in recombinants IS127CTX-M-3 and 127CTX-M-3, which contained a 127 bp spacer with or without ISEcp1, respectively. In conclusion, the promoter within ISEcp1 and a shorter spacer (42 bp but not 127 bp) between ISEcp1 and blaCTX-M are necessary for high-level blaCTX-M-3 expression.