Macrocyclic aminoacyl-AMP analogs have been developed to inhibit non-ribosomal peptide synthetase amino acid adenylation domains selectively by mimicking a cisoid ligand binding conformation observed in crystal structures. In contrast, these macrocycles do not inhibit aminoacyl-tRNA synthetases, which are mechanistically closely related but bind their ligands in a distinct transoid conformation. The macrocycles contain a two- or three-carbon linker between Cβ of the amino acid moiety and C8 of the adenine ring and a sulfamate in place of the phosphate group. These compounds are potent inhibitors of the cysteine adenylation domain activity of the yersiniabactin siderophore synthetase HMWP2 and, unlike the corresponding linear aminoacyl-AMP analogs, do not inhibit protein translation in vitro. Selective small molecule inhibitors of non-ribosomal peptide synthesis should provide a powerful means to study the biological functions of non-ribosomal peptide natural products and a potential avenue to develop novel antibiotics.