Compound 1 was isolated and purified from the fermentation broth of a marine sediment-derived Bacillus amyloliquefaciens SH-B74. Its structure was characterized by one-dimensional and two-dimensional NMR spectroscopy as well as tandem mass spectrometry (MS/MS) analysis. HRESI MS showed m/z 1057.5177 ([M+Na]+) corresponding to the chemical formula C47H74N10NaO16. NMR data revealed 12 N-bonded protons, a para-substituted benzene ring, seven α-protons of peptide bonds, a long fatty-acid chain, and characteristic signals of a β-amino fatty-acid residue. MS/MS confirmed the sequence of Asn1, C15 β-amino fatty-acid residue2, Thr3, Ser4, Glu5, Ser6, Asn7, and Tyr8. Compound 1 was identified as a new lipopeptide antibiotic belonging to the bacillopeptin family, named bacillopeptin B1. It exhibited antagonistic activities against plant fungal pathogens such as Valsa mali, Fusarium oxysporum f.sp. cucumerinum, and Rhizoctonia solani when the concentration was over 2 mg/ml.