The nutritional requirements for the maximum production of lipopeptides by Bacillus subtilis N7 (B. subtilis N7) were investigated and optimized using response surface methodology (RSM) under shake flask fermentation. A one-factor-at-a time experimental setup was used to screen carbon and nitrogen sources. A Plackett?Burman design (PBD) was employed to screen the most critical variables for lipopeptides production amongst ten nutritional elements. The central composite experimental design (CCD) was finally adopted to elucidate the composition of the fermentation medium. Statistical analyses (analysis of variance, ANOVA) of the results showed that KCl, MnSO4 and FeSO4·6H2O were important components and that their interactions were strong. Lipopeptide production was predicted to reach 709.87 mg/L after a 60 h incubation using an optimum fermentation medium composed of glucose 7.5 g/L, peanut oil 1.25 g/L, MgSO4 0.37 g/L, KH2PO4 0.75 g/L, monosodium glutamate 6.75 g/L, yeast extract and NH4Cl (5:3 w/w) 10 g/L, KCl 0.16 g/L, FeSO4·6H2O 0.24 mg/L, MnSO4 0.76 mg/L, and an initial pH of 7.0. Lipopeptide production (706.57 ± 3.70 mg/L) in the optimized medium confirmed the validity of the predicted model.