Objective To reveal the influence factor of ampD specific mutation on the transformation of AmpC β-lactamase from non-high-productive type to derepressed the high-productive type in Enterobacter cloacae. Methods The selected cbromatin-mediated derepressed high-productive strains of Enterobacter cloacae and genomic DNA were extracted, and genomic ampD was amplified by using PCR, and then sequenced and confirmed by specific mutation. Use site-directed mutagenesis to mutate the wild-type Enterobacter cloacae for the above site. The changes of AmpCβ-lactamase types were detected by the improved Cefotaxime three-dimensional test. Results Of the 121 strains of Enterobacter cloacae,15 strains were ehromatin-mediated derepressed high-productive type,8 strains had significant mutations and 7 strains of site-directed mutagenesis were established. Ecl MA(274 inser-ting A)、Ecl MC(327 missing C)and Ecl MF(27 inserting G)were changed from non-high-productive type to derepressed-high-pro-ductive type,but Ecl MB(371 inserting T), Ecl MD(515 missing C), Ecl ME(324 C→A), Ecl MG(238C→A 302T→A)did not change in AmpC β-lactamase types. Conclusion The significant mutations that could lead to the transformation of AmpC β-lacta-mase were the Frame-shift mutations that could create the change of large region of amino acid. However, other mechanisms for the derepressed high-productive AmpC β-lactamase were not excluded in the study.