21-Hydroxyoligomycin A (1) was isolated from Streptomyces cyaneogriseus ssp. noncyanogenus (LL-F28249) and fully characterized by NMR and single-crystal X-ray diffraction methods. The complete 1H and 13C NMR chemical shift assignments for 1 were made using 2D NMR experiments, and the chirality at C-21 was deduced to be R from a J-based configuration analysis. The absolute configuration at C-21 and at the other 18 chiral centers in the molecule were independently confirmed by anomalous dispersion measurements on a crystal of the chloroform methanol solvate of 21-hydroxyoligomycin A (1).