The present study investigated the stimulation effect of L-glutamine (L-gln) on bacillomycin D biosynthesis of Bacillus subtilis fmbJ and the transcription of the synthetase genes and the signal proteins that related to the biosynthesis of bacillomycin D. It was found that L-gln significantly increased the production of bacillomycin D, in comparison with other amino acids. L-gln significantly enhanced the expression level of bmyA (BYA), bmyB (BYB), bmyC (BYC), the thioesterase gene (TE) and up-regulated DegU, o(H) and SpoOA, but down-regulated AbrB, therefore the yield of bacillomycin D significantly increased. Furthermore, when conjointly feeding 5 g/L of L-gin and 30 g/L of inulin, the production of bacillomycin D was up to 1.93 g/L.