The structure determination of the 30-membered cyclodepsipeptide decatransin was demonstrated on the basis of total synthesis. Both (R)- and (S)-2-hydroxy-5-methylhexanoic acid derivatives were prepared via the Evans asymmetric alkylation. N-Alkyl-enriched peptide fragments were synthesized by the Cbz strategy in the solution phase without formation of diketopiperazine and epimerization. The synthesis of putative candidates was achieved by convergent peptide coupling of three peptide fragments, followed by macrocyclization under the Mitsunobu conditions.