<jats:title>ABSTRACT</jats:title><jats:p>We identified the biosynthetic gene clusters of the siderophore nocobactin NA. The<jats:italic>nbt</jats:italic>clusters, which were discovered as genes highly homologous to the mycobactin biosynthesis genes by the genomic sequencing of<jats:italic>Nocardia farcinica</jats:italic>IFM 10152, consist of 10 genes separately located at two genomic regions. The gene organization of the<jats:italic>nbt</jats:italic>clusters and the predicted functions of the<jats:italic>nbt</jats:italic>genes, particularly the cyclization and epimerization domains, were in good agreement with the chemical structure of nocobactin NA. Disruptions of the<jats:italic>nbtA</jats:italic>and<jats:italic>nbtE</jats:italic>genes, respectively, reduced and abolished the productivity of nocobactin NA. The heterologous expression of the<jats:italic>nbtS</jats:italic>gene revealed that this gene encoded a salicylate synthase. These results indicate that the<jats:italic>nbt</jats:italic>clusters are responsible for the biosynthesis of nocobactin NA. We also found putative IdeR-binding sequences upstream of the<jats:italic>nbtA</jats:italic>, -<jats:italic>G</jats:italic>, -<jats:italic>H</jats:italic>, -<jats:italic>S</jats:italic>, and -<jats:italic>T</jats:italic>genes, whose expression was more than 10-fold higher in the low-iron condition than in the high-iron condition. These results suggest that<jats:italic>nbt</jats:italic>genes are regulated coordinately by IdeR protein in an iron-dependent manner. The Δ<jats:italic>nbtE</jats:italic>mutant was found to be impaired in cytotoxicity against J774A.1 cells, suggesting that nocobactin NA production is required for virulence of<jats:italic>N. farcinica</jats:italic>.