During industrial fermentation, Streptomyces clavuligerus F613-1 simultaneously produces primary product clavulanic acid (CA) and cephamycin C. The cephamycin C biosynthetic gene cluster and pathway have been basically elucidated and the CcaR positive regulator was found to control the cephamycin genes expression. However, additional mechanisms of regulation cannot be excluded. The BB341_RS13780/13785 gene pair in S. clavuligerus F613-1 (annotated as SCLAV_2960/2959 in S. clavuligerus ATCC27064) encodes a bacterial two-component system (TCS) and were designated as CepRS (for cephamycin regulator/sensor). CepRS significantly affects cephamycin C production but only slightly affects CA production. To further understand the regulation of cephamycin C biosynthesis, the cepRS genes were deleted from S. clavuligerus F613-1. The deletion mutant resulted in decreased cephamycin C production but had no phenotypic effects. Real-time quantitative polymerase chain reaction analysis revealed that CepRS regulates the expression of most genes involved in cephamycin C biosynthesis, with electrophoretic mobility shift assays showing that CepR interacts with the cefD-cmcI intergenic region. These results demonstrate that the CepR response regulator serves as a transcriptional activator of cephamycin C biosynthesis, which may provide an approach for metabolic engineering methods for CA production by S. clavuligerus F613-1 in future.