The structure and absolute configuration of piperazinomycin have been established by X-ray crystallographic analysis of its monohydrobromide (II, C₁₈H₂₀N₂O₂·HBr). Piperazinomycin monohydrobromide crystallizes in the orthorhombic system with space group P2₁2₁2₁ (a=8.926(5), b=22.341(11), c=8.159(2) Å, Z=4). The structure was solved by the heavy atom method and successive difference Fourier synthesis, refined by least-squares methods (final R factor=0.083), and the absolute configuration was determined via the anomalous dispersion method, confirming the S configuration at both C(2) and C(2') chiral centers. Piperazinomycin possesses a unique cyclic structure where two benzene rings and one piperazine ring are linked by O(1'), C(3), and C(3'). The piperazine ring adopts an almost ideal chair conformation, with the two benzene rings nearly perpendicular (dihedral angle=80.4°). Intramolecular hydrogen bonding exists between the hydroxyl group O(1)H and O(1') of the diphenyl ether, while the bromide anion forms intermolecular hydrogen bonds contributing to a three-dimensional network. Biogenetically, piperazinomycin is assumed to be derived from two molecules of L-tyrosine, consistent with its absolute configuration and relationship to the tyrosine-derived metabolite herquline.