Sulfur-containing peptide antibiotic siomycin (SIM) isolated from Streptomyces sioyaensis belongs to the thiostrepton group of antibiotics and consists of one major component (SIM-A) and two minor components (SIM-B and -C). The structures of SIM-A, -B, and -C were previously elucidated. Further search for other minor components led to the isolation of SIM-D1. Here we report the isolation of SIM-D1 (II) and its structure elucidation by 1H and 13C NMR spectroscopy. SIM-D1 was separated from siomycin complex by silica gel column chromatography using a CHCl3-CH3OH mixture and purified by repeated column chromatography or preparative thin-layer chromatography. It has physicochemical properties (mp ca. 260°C decomp., [α]D-69.9°, Rf 0.13, UV, IR, amino acid analysis) similar to SIM-A and exhibits in vitro antibacterial activity comparable to SIM-A against Gram-positive bacteria. 1H NMR spectroscopy of SIM-D1 in CDCl3 showed the absence of signals for Q-12 CH3 (δH 1.37) and Q-11 CH (δH 5.34) (present in SIM-A) and the presence of an ABX pattern assignable to a CH2OH grouping (around δH 4.5 and 5.0), which changed to an AX-type signal upon D2O addition. 13C NMR spectroscopy confirmed the substitution of the CH(OH)CH3 group in the Q residue of SIM-A with a CH2OH group in SIM-D1: SIM-D1 exhibited a signal at δC 61.7 (Q-11 CH2OH) instead of signals at δC 65.1 (Q-11 CH) and 23.2 (Q-12 CH3) in SIM-A. Based on these NMR data, the structure of SIM-D1 was determined.