In this study we report the isolation, structure elucidation, and biosynthesis of mirubactin (1), a siderophore containing an unprecedented chemical functionality in natural products, namely, an O-acyl hydroxamic acid ester. Mirubactin represents the first siderophore isolated from the genus Actinosynnema and the first natural product produced by Actinosynnema mirum whose biosynthetic gene cluster could be identified. Structure elucidation was accomplished through a combination of spectroscopic (NMR, IR, and UV/vis) and mass spectrometric methods and revealed the presence of an unusual ester bond between the δ-N-hydroxyl group of δ-N-formyl-δ-N-hydroxyornithine and a 2,3-dihydroxybenzoate moiety. Bioinformatic analysis of the A. mirum genome and subsequent biochemical characterization of the putative biosynthetic machinery identified the gene cluster responsible for mirubactin assembly. The proposed biosynthesis of mirubactin comprises the iterative use of a stand-alone carrier-protein-bound substrate, as well as an ester-bond-forming step catalyzed by a C-terminal condensation domain, thus revealing an interesting system for further biochemical studies to gain a deeper understanding of nonribosomal peptide synthetase-catalyzed siderophore biosynthesis.