Objective To explore the RecET-Cre/loxPsystem for chromosomal replacement of promoter and its application on enhancementl-leucine production inCorynebacterium glutamicum(C. glutamicum) ATCC14067. Results The RecET-Cre/loxPsystem was used to achieve the chromosomal replacement of promoter inC. glutamicumATCC14067 to adjust the metabolic flux involving thel-leucine synthetic pathway. First,leuA(R)_13032 fromC. glutamicumATCC13032 which carried two mutations was overexpressed to release enzyme feedback inhibition. Then, comparing different mutations inilvBNCgene clusters, the results indicated thatilvBNC_CP was most effective to enhance the metabolic flux of pyruvate towardsl-leucine synthesis. The promoters ofpck, odxandpyk2 were overexpressed under the strong promoter Peftuor Psodto improve the supply of pyruvate. Besides, the promoter PilvBNCwas employed to dynamically control the transcription level oficddue to its attenuation mechanism by responding to the concentration ofl-leucine. The final engineered strain produced 14.05 gl-leucine/L in flask cultivation. Conclusion The RecET-Cre/loxPsystem is effective for gene manipulation inC. glutamicumATCC14067. Besides, the results demonstrate the potential ofC. glutamicumATCC14067 forl-leucine production and provide new targets and strategies for strain development.