The effect of inulin on the production of bacillomycin D and the levels of mRNA of bacillomycin D synthetase genes: bmyA (BYA), bmyB (BYB), bmyC (BYC), the thioesterase gene (TE) and regulating genes: AbrB, ComA, DegU, PhrC, SigmaH and Spo0A in Bacillus subtilis fmbJ were investigated. The production of bacillomycin D was enhanced with the increase of biomass concentration. The maximum production and productivity of bacillomycin D were found to be 1227.49 mg/L and 10.23 mg/L h. Inulin significantly improved the expression of bacillomycin D synthetase genes: bmyA (BYA), bmyB (BYB), bmyC (BYC) and the thioesterase gene (TE). Also, inulin up-regulated ComA, DegU, SigmaH and Spo0A and therefore promoted the high production of bacillomycin D. Our results provided a practical approach for efficient production of bacillomycin D and a meaningful explanation for regulatory mechanism of bacillomycin D biosynthesis.