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Summary: Edeines, a group of cationic antimicrobial peptides produced by the soil bacterium Brevibacillus, have broad biological effects, such as antimicrobial, anticancer and immunosuppressive activities. However, the yield of edeines in wild‐type (WT) Brevibacillus is extremely low, and chemical synthesis of edeines is a time‐consuming process. Genetic engineering has proven to be an effective approach to produce antibiotics with high yield. In this study, the edeine biosynthetic gene cluster (ede BGC), which is involved in edeine production, was identified and characterized in Brevibacillus brevis X23. To improve edeine production in B. brevis X23, the ede BGC promoter was replaced with six different promoters, Pmwp, Pspc, PxylA, Pshuttle‐09, Pgrac or P43, through double‐crossover homologous recombination. The new promoters significantly increased the expression of the ede BGC as well as edeine production by 2.9 ± 0.4 to 20.5 ± 1.2‐fold and 3.6 ± 0.1to 8.7 ± 0.7‐fold respectively. The highest yield of edeines (83.6 mg l−1) was obtained in B. brevis X23 with the Pmwp promoter. This study provides a practical approach for producing high yields of edeines in B. brevis. [ABSTRACT FROM AUTHOR]