Bacilohydrin　A,　a　new　cyclic　lipopeptide　composed　of　seven　mixed　(D/L)-amino　acids　((L)-Ile(L)(1)-Leu(D)(2)-Asp(L)(3)-Val(L)(4)-Leu(L)(5)-Leu(L)(6)-Gln(7))　and　a　(R)-3-hydroxy-12-methyltetradecanoic　acid　(HMT)　residue,　was　isolated　from　the　crude　culture　extract　of　Bacillus　sp.　SY27F,　which　was　obtained　from　the　Indian　Ocean　hydrothermal　vent.　Its　planar　structure　was　elucidated　by　spectroscopic　analyses,　including　1D　and　2D　NMR　spectroscopy,　mass　spectrometry　(MS),　and　secondary　ion　mass　spectrometry　(MS/MS),　whereas　its　absolute　configuration　was　determined　by　modified　Marfey＇s　analysis.　Bacilohydrin　A　displayed　significant　cytotoxicities　against　DU-145,　MCF-7　and　HepG2　cancer　cell　lines　with　IC50　values　of　50.3-175.1　nM.　Additionally,　this　is　the　first　time　to　determine　the　absolute　configuration　of　a　D-aspartic　acid　residue　in　surfactin　class.