The new siderophore tsukubachelin B (1) was isolated from the iron-deficient culture medium of the newly isolated strain Streptomyces sp. TM-74. The chemical structure of tsukubachelin B (1) was established via interpretation of 2D nuclear magnetic resonance and electrospray ionization-mass spectroscopic data. The structure of tsukubachelin B (1) comprises 6 mol of amino acids, including 2 mol of serine and 1 mol each of ornithine, N-alpha-methyl-N-delta-hydroxy-N-delta-formylornithine, N-alpha-methyl-N-delta-hydroxyornithine, and cyclic N-hydroxyornithine.