The lipopeptide samples were purified by normal reverse-phase column chromatography after isolated from the cell free broth of Brevibacillus brevis HOB1. A lipopeptide was separated from these samples by preparative HPLC, and determined to have a molecular weight of 1035.7 D by ESI-MS. The fatty acid part was identified to be C15 β-hydroxyl fatty acid by GC/MS. A quantitative analysis of the PITC-derivated amino acids of the lipopeptide by HPLC gave the following molar ratios: Asp 1, Glu 1, Val 1, Leu 4. All the results indicated that the lipopeptide has a structure similar to C15 surfactin. This study revealed that surfactin series lipopeptides can be produced by Brevibacillus genus in addition to Bacillus genus.