The total synthesis of the piperazic acid-containing antibiotic cyclic peptide, hytramycin V, has been achieved. Unexpected cleavage of the peptide bond was observed during the synthesis of a pentapeptide, we then successfully found that the addition of 2,6-di-tert-butylpyridine (2,6-DTBP) was effective to prevent the cleavage upon acylation with AgCN, leading to a pentapeptide in excellent yield. The synthesis of a hexapeptide, followed by global deprotection of the protecting groups provided a cyclization precursor. Finally, macrolactamization of the precursor using T3P®under high-dilution conditions furnished the desired natural product, hytramycin V. The synthesis of the enantiomer of hytramycin V was also achieved, and no difference between the enantiomers was observed in the evaluation of their antibacterial activity against Mycobacteriumstrains, revealing the fact that the potency of the activity was not dependent on the chirality of the cyclopeptide backbone.We successfully achieved the total synthesis of hytramycin V through AgCN-catalyzed acylation in the presence of 2,6-di-tert-butylpyridine (2,6-DTBP) and macrolactamization using T3P®under high-dilution conditions. Synthesis of the enantiomer of hytramycin V was also achieved, and no difference between the enantiomers was observed in the evaluation of an antibacterial activity against Mycobacteriumstrains.