AbstractA kinetic model describing the cephalosporin biosynthesis in Streptomyces clavuligerus was developed. Using previously reported kinetic data of biosynthetic enzymes, we examined the kinetics of cephalosporin production. The predicted time profile of the specific production rate during a batch culture parallels that of experimental observation. Sensitivity analysis reveals that δ‐(L‐α‐aminoadipyl)‐L‐cysteinyl‐D‐valine (ACV) synthetase is the rate‐limiting enzyme. The effect of amplifying ACV synthetase on the specific production rate was analyzed theoretically. Increasing ACV synthetase enhances the production rate initially until ACV synthetase enhances the production rate initially until deacetocycephalosporin C hydroxylase becomes rate‐limiting. Such kinetic analysis can provide a rational basis for modifying the biosynthetic machinery of cephalosporin through gene cloning.