<jats:title>Abstract</jats:title><jats:p>Salinamide A belongs to a rare class of bicyclic depsipeptide antibiotics in which the installation of a (4‐methylhexa‐2,4‐dienoyl)glycine handle across a hexadepsipeptide core contributes to its chemical complexity and biological properties. Herein, we report the genetic and biochemical basis for salinamide construction in the marine bacterium <jats:italic>Streptomyces</jats:italic> sp. CNB‐091, which involves a novel intermolecular transesterification reaction catalyzed by a type I thioesterase. Heterologous expression studies revealed the central role of the nonribosomal peptide synthetase Sln9 in constructing and installing the distinctive acylglycine “basket handle” of salinamide. Biochemical characterization of the Sln9 thioesterase domain established that transesterification of the serine residue of desmethylsalinamide E with acylated glycyl thioesters yields desmethylsalinamide C.