The total synthesis of surfactin B2, a cyclic depsipeptide isolated from Bacillus natto KMD 2311, was achieved to elucidate the absolute configuration of its fatty acid moiety. This is the first chemical confirmation of the absolute configuration of a surfactin homolog. Two possible diastereoisomers of surfactin B2, cyclo[D- and L-3-(Glu-Leu-D-Leu-Val-Asp-D-Leu-Leu-O)-n-tetradecanoyl] (1a and b), were synthesized by a solution method using mainly active ester and azide fragment condensation methods. Cyclization reaction of the partially protected linear depsipeptide containing the C-terminal N-succinimidyl active ester in pyridine by the high dilution method at room temperature for 3d gave the desired cyclic depsipeptide in a high yield of about 70%. The synthetic product 1a, containing the D-isomer of 3-hydroxytetradecanoic acid as a fatty acid moiety, was identical with natural surfactin B2.