Three new cyclic lipopeptides (compounds 1-3) were isolated from Bacillus sp. SY27F, which collected from the deep-sea hydrothermal vent in the Indian Ocean. The planar structures of compounds 1-3 were characterized by LC-MS, GC-MS and NMR, whereas their absolute configurations were determined by modified Marfey's analysis and optical rotation contrast. They shared the same heptapeptide chain (N-L-Gln(1)-D-Leu(2)-L-Leu(3)-L-Val(4)-L-Asp(5)-D-Leu(6)-L-Ile(7)-C), differing only by the length of the fatty acid chain. Compounds 1-3 displayed significant cytotoxic activity against DU145, MCF-7 and HepG2 cancer cell lines with IC50 values of 96.8-190.7 nM and moderate to weak cytotoxicity against A549, HeLa and HT1080, with IC50 values of 9.3-43.2 mu M, respectively. However, none of the three compounds showed any antibacterial activity against Escherichia coli, Staphylococcus aureus and Monilia albicans.