<jats:title>Abstract</jats:title><jats:p>A new lipopeptide with C<jats:sub>12</jats:sub>fatty acid has been isolated from the cell broth of<jats:italic>Bacillus subtilis</jats:italic>HSO121 by chromatographic methods, which is believed to be the homologue of lipopeptides. The fatty acid portion was methylated and analyzed by GC/MS, ESI Q‐TOF MS and<jats:sup>1</jats:sup>H‐NMR. The peptide portion, of which the amino acid composition was obtained by HPLC combined with a phenyl isothiocyanate (PITC) derivatization methods, was analyzed by ESI Q‐TOF MS. Comparing the obtained results with surfactin C<jats:sub>13</jats:sub>showed that the new lipopeptide has a peptide moiety similar to that of surfactin and the difference exists in the fatty acid portion, which is an iso‐C<jats:sub>12</jats:sub>β‐hydroxy fatty acid. The critical micelle concentration (CMC) of this new homologue is estimated to be 6.27 × 10<jats:sup>−5</jats:sup>mol/l in 10 mmol/l phosphate buffer solution (PBS, pH 8.0) at 30 °C, and the surface tension at CMC (γ<jats:sub>CMC</jats:sub>) achieved is as little as 27.71 mN/m. The hemolytic activities of the C<jats:sub>12</jats:sub>‐lipopeptide on 2% human erythrocytes showed a HC<jats:sub>50</jats:sub>of 26.5 µmol/l. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.