The DNA binding properties of three cation-substituted anthrapyrazole derivatives of emodin with calf thymus DNA were characterized by spectroscopic methods and the specific binding modes were elucidated. At low drug and high DNA concentrations, compound 1 with a mono-cationic amino side chain exhibited an intercalative binding mode, 2 with a much longer and more flexible di-cationic side chain exhibited an external binding mode, and 3 with a rigid di-cationic side chain exhibited both intercalative and external binding modes. The DNA binding mode of compounds was altered after structural modification. The molecular structure-DNA binding relationships found from this study may be useful for the design of anthrapyrazole derivatives with desired binding characteristics.