This study aimed to improve the efficiency of bioconversion of cephalosporin C to 7α-methoxycephalosporin C. We performed the genetic modulation of the expression level of tailoring genes cmcI and cmcJ, which are responsible for the methoxylation of cephalosporin C, to improve the efficiency of bioconversion in the recombinant strains. The results showed that the coordinate adjustment of the enzyme amounts of CmcI and CmcJ in the host strain by placing the ermEp* promoter in the upstream of both cmcI and cmcJ genes in their expression cassette can improve the efficiency of bioconversion from cephalosporin C to 7α-methoxycephalosporin C. Under the optimized cultivation condition, 2·0 g l(-1) of the substrate cephalosporin C can be transformed into 0·78 g l(-1) of 7α-methoxycephalosporin C by the extract of the recombinant strain Streptomyces clavuligerus DYG3003, in which the transcript ratio of cmcJ/cmcI has been engineered to 1·7 : 1. Coordinate adjustment of the enzyme amounts of CmcI and CmcJ in the host strain can greatly enhance the efficiency of bioconversion from cephalosporin C (CPC) to 7a-methoxycephalosporin (MCPC). As one kind of the most important β-lactam antibiotics, 7α-methoxycephalosporins show stronger activity against penicillin-resistant bacteria than cephalosporins because of the higher stability of 7α-methoxycephalosporins to β-lactamase. Our study provides a practical strategy to perform the industrial production of the clinically useful 7α-methoxycephalosporin C through biotransformation.