The lipopeptide and its homologues are a kind of the five major biosurfactants with prominent interfacial and biological activities. A suite of mutagenesis method was adopted to expose a wild lipopeptide-producing strain Bacillus subtilis HSO121 to improve lipopeptide yield, and a stable mutant named R2-104 with a 2.0-fold production of lipopeptide was obtained. Compared to that of the wild strain HSO121, the lipopeptide produced by R2-104 showed a similar surface activity, but the course profiles of lipopeptide production during cultivation were different, with the peak yield of 500 mg at about 9 h by R2-104, and 400 mg at about 5 h by HSO121. The constituent abundance of the lipopeptide homologues produced by R2-104 was also different from that by HSO121. Combined methods of ESI-MS, GC-MS and MS-MS were applied for structural characterization of lipopeptide homologues, and it showed that the lipopeptides produced by R2-104 and HSO121 were attributed to a surfactin family with different constituents. The dominant constituent of the surfactin family produced by R2-104 was anteiso C-15-surfactin with a relative content of 43.8 %, while the dominant one produced by HSO121was iso C-14-surfactin with a relative content of 33.1 %.