Some Clostridium difficile strains produce, in addition to toxins A and B, the binary toxin Clostridium difficile transferase (CDT), which ADP-ribosylates actin and may contribute to the hypervirulence of these strains. The separate binding and translocation component CDTb mediates transport of the enzyme component CDTa into mammalian target cells. CDTb binds to its receptor on the cell surface, CDTa assembles and CDTb/CDTa complexes are internalised. In acidic endosomes, CDTb mediates the delivery of CDTa into the cytosol, most likely by forming a translocation pore in endosomal membranes. We demonstrate that a seven-fold symmetrical positively charged β-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-β-cyclodextrin, which was developed earlier as a potent inhibitor of the translocation pores of related binary toxins of Bacillus anthracis, Clostridium botulinum and Clostridium perfringens, protects cells from intoxication with CDT. The pore blocker did not interfere with the CDTa-catalyzed ADP-ribosylation of actin or toxin binding to Vero cells but inhibited the pH-dependent membrane translocation of CDTa into the cytosol. In conclusion, the cationic β-cyclodextrin could serve as the lead compound in a development of novel pharmacological strategies against the CDT-producing strains of C. difficile.