Polytheonamide B (1) is a potent cytotoxic natural product isolated from marine sponge and is by far the largest non-ribosomal peptide known to date. The 48 amino acid residues including a variety of non-proteinogenic amino acids have the sequence of alternating D- and L-chirality. These structural features permit the formation of a stable β-strand-type structure with all of the side chains on one side, thereby encouraging the formation of a helix (6.5 residues per turn). In the biological setting, cations can be transported across lipid bilayer of cells through the pore to exert its bioactivity. The unique and synthetically challenging structure of 1 together with the potent cytotoxicity motivated us to launch a project for their chemical construction. Here we report the first total synthesis of polytheonamide B The containing non-proteinogenic amino acids of 1 were prepared by multi-step syntheses from readily available starting materials. Then, four peptide fragments {Ncap-[1-11]-OH (A), Fmoc-[12-25]-OH (B), Fmoc-[26-32]-OH (C), Fmoc-[33-48]-OH} (D) were synthesized on solid phase under the carefully optimized conditions. Fragments A, B and C were derivatized to the corresponding thioester, and the union of these fragments was performed using AgNO_3 in the presence of HOOBt in a stepwise fashion, delivering the protected polytheonamide B. The last global deprotection was realized under the acidic conditions to provide polytheonamide B (1).