Clostridium perfringens epsilon toxin (Etx) is an extremely potent toxin, causing fatal enterotoxaemia in many animals. Several amino acids in domains I and II have been proposed to be critical for Etx to interact with MDCK cells. However, the critical amino acids in domain III remain undefined. Therefore, we assessed the effects of aromatic amino acids in domain III on Etx activity in this study. All of the results indicated that Y71 was critical for the cytotoxic activity of Etx towards MDCK cells, and this activity was dependent on the existence of an aromatic ring residue in position 71. Additionally, mutations in Y71 did not affect the binding of Etx to MDCK cells, indicating that Y71 is not a receptor binding site for Etx. In summary, we identified an amino acid in domain III that is important for the cytotoxic activity of Etx, thereby providing information on the structure-function relationship of Etx.