Background: New Delhi metallo-beta-lactamase 1 (NDM-1) is considered to be an important factor of antimicrobial resistance in Enterobacteriaceae. In China, the bla(NDM-1) gene has been mostly detected in carbapenem-resistant Acinetobacter spp. but is less reported in Enterobacteriaceae and more rarely found in E. cloacae. Objectives: This study explored the genetic features of the bla(NDM-1) gene of E. cloacae and a bla(NDM-1) knockout mutant was constructed using Red homologous recombination. In addition, the effect of the knockout on antimicrobial resistance, growth ability, and in vitro competitiveness was investigated. Methods: The upstream and downstream structures of the bla(NDM-1) gene were analyzed in ten E. cloacae isolates using primer walking and PCR mapping. A bla(NDM-1) knockout mutant was constructed through Red homologous recombination and verified by PCR, RT-qPCR, and sequencing. The antimicrobial susceptibility, growth curves, and in vitro growth competitiveness were compared between the bla(NDM-1 )knockout mutant and the parental strain. Results: All E. cloacae study isolates except for strain T10, contained an identical bla(NDM-1) gene structure. The Delta ISAba125 truncated by ISEc33 element and the bleo followed by a Delta trpF and ISSen4 was located immediately upstream and downstream of T1-T9 strains. However, the Delta ISAba125 and the bleo followed by a Delta trpF were located immediately upstream and downstream, respectively, in the TR) strain. PCR, RT-qPCR, and DNA sequencing analyses showed that the bla(NDM-1) knockout mutant was successfully constructed. The bla(NDM-1) knockout mutant and the parental strain exhibited similar resistance patterns to penicillin, cephalosporins, aminoglycosides, tetracycline, and quinolones. Both strains displayed similar growth curves in Luria Broth. The competition index (CI), defined as the knockout mutant/parental strain ratio was 0.69 in the competition experiment in vitro. Conclusions: The DNA regions upstream and downstream of the bla(NDM-1) gene often contained insertion sequences and elements. Red homologous recombination was successfully used to knock out bla(NDM-1) in E. cloacae, which allowed us to decipher the links between this gene, antimicrobial resistance, and bacterial growth competitiveness.