Surfactin is a peptidelipid consisting of a fatty acid and a heptapeptide with the sequence of L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-LeuOH. This study aimed to determine the structure of the fatty acid in surfactin, its linking site to the peptide, and elucidate the total structure of surfactin. The fatty acid was recovered from surfactin as its methyl ester and dimethyl N-acyl glutamate via methanolysis. Through silica gel-thin layer chromatography, elemental analysis, IR spectrum, NMR spectrum, mass spectrometry, and an experiment where surfactin was hydrolyzed in 6 N HCl followed by hydrogenation of the petroleum ether-soluble fraction, the fatty acid was identified as 3-hydroxy-13-methyl-tetradecanoic acid. The carboxyl group of the fatty acid was found to form an amide bond with the amino group of L-Glu in the peptide, as confirmed by the isolation of dimethyl N-acyl glutamate. Thus, the total structure of surfactin was elucidated.