The first synthesis of the anti‐TB cyclic peptide callyaerin A containing a rare (Z)‐2,3‐diaminoacrylamide bridging motif is reported. Fmoc‐formylglycine‐diethylacetal was used as a masked equivalent of formylglycine in the synthesis of the linear precursor of callyaerin A. Intramolecular cyclization between the formylglycine residue and the N‐terminal amine in the linear peptide precursor afforded the macrocyclic natural product callyaerin A. Synthetic callyaerin A possessed potent anti‐TB activity (MIC100 = 32 µM) while its all‐amide congener was inactive. Variable temperature NMR studies of both the natural product and its all amide analogue revealed the extraordinary rigidity imposed by this diaminoacrylamide unit on peptide conformation. The work reported herein pinpoints the intrinsic role that the rare (Z)‐2,3‐diaminoacrylamide moiety confers on peptide bioactivity.