A method for the separation and preparation of validamycin A and validamycin B by anion-exchange resin is described. The two compounds, validamycin A and validamycin B, could be separated effectively in the 10 x 400 mm column with anion-exchange resin A600 under the optimized conditions: elution flow rate of 0.25 mL/min, added quantity of 0.7 g, bed height of 330 mm, and deionized water as eluent. The yields of validamycin A and validamycin B were 58.9% and 89.5%, respectively.