Gymnopeptides A and B are unprecedented highly N-methylated cyclic β-hairpin octadecapeptides with striking antiproliferative activities isolated from the mushroom Gymnopus fusipes. Using Fmoc-based solid-phase peptide synthesis, followed by macrolactamization of the resulting linear peptides, the first total synthesis of gymnopeptides A and B was successfully achieved in this study. The coupling methods used for the solid-phase synthesis and the cyclization were optimized, and the configuration of the Ser1/Thr1 residue in gymnopeptide A/B was determined to be l.