Abstract: Objective: This paper mainly studied the effects and regulation of exogenous addition of pyruvate, overexpression of pyruvate kinase and acetyl-CoA synthase on the synthesis of Bacillomycin D. Methods: In this study, B. amyloliquefaciens fmbJ was used as the initial strain. Firstly, the effect of exogenous pyruvate with different concentrations on the yield of Bacillomycin D was detected by HPLC. The regulation of pyruvate in this process was detected by RT-PCR. Then, pyruvate kinase (pyk) and acetyl-CoA synthase (acs) overexpression strains B. amyloliquefaciens fmbJ-pyk and fmbJ-acs were constructed by electrotransformation, and their Bacillomycin D production induced by different concentrations of IPTG was studied. Results: 0.075% pyruvate could increase the yield on Bacillomycin D to 1.41-fold that of the control group. The upregulation of Bacillomycin D synthesis gene also verified this enhancement. In addition, pyruvate could up-regulate the expression of regulatory factors such as comA, comP, comQ, sigH, sigM, degU, degQ, spo0A and codY, down-regulate the expression of rapC and abrB, promote the production of acetyl CoA synthase, and finally jointly promote the synthesis of Bacillomycin D. Under the induction of 50 mg/L IPTG, the yield of Bacillomycin D of fmbJ-acs and fmbJ-pyk increased to 1.16-fold and 1.34-fold that of fmbJ, respectively. Conclusion: This study found that exogenous addition of pyruvate and overexpression of acs or pyk could promote the synthesis of Bacillomycin D, clarified the regulation of pyruvate in the synthesis of Bacillomycin D, and widened a new research idea for promoting the efficient production of Bacillomycin D.