Isolation and purification of lipopeptides from Bacillus subtilis fmbR was studied in this paper. With HPLC methods, it was determined that the antimicrobial substance was composed of many components, and one of tham had the similar retaining time to surfactin. In addition, the antimicrobial substance was proved to include the closed circle peptide band by TLC, and one of them had the similar moving rate to surfactin. Furthermore, ESI-MS analysis showed that the antimicrobial substance contained three homologues of surfactin, such as m/z1009.1,m/z1023.2 and m/z1037.0. MALDI-TOF-MS analysis indecated it also contained the [M+H]+ ion peak at m/z 3403.95, which is very similar to molecular mass of subtilosin produced by Bacillus subtilis168.