Clostridium difficile is a bacterial pathogen that causes serious and potentially fatal inflammatory disease of the colon. Anti-virulence strategies to treat C. difficile infection are attractive options to antibiotic therapy because the human microbiota is spared and damage to the host tissue is minimized. TcdA and TcdB are the major virulence factors produced by C. difficile and mediate host cytotoxicity by glycosylating and inactivating Rho GTPases using UDP-glucose as a glycosyl donor, leading to cell rounding, cell death and loss of intestinal integrity. We used kinetic isotope effects (KIEs) and transition state (TS) theory to solve the TS structure of the glycosyltransferase domain of TcdA and TcdB. This permits the design of TS analogues which are powerful enzyme inhibitors. KIE analysis supported the formation of a dissociative glucocation TS where positive charge develops on the anomeric carbon. Iminosugars mimic glucocationic TS’s as they contain cationic nitrogen groups at or near the anomeric carbon. We identified iminosugars, isofagomine and noeuromycin as TS analogue inhibitors of TcdA and TcdB and we characterized them by kinetic, thermodynamic and structural analysis. Both iminosugars exhibit nM binding constants and inhibit TcdA and TcdB by forming ternary complexes with UDP, a product of the glycosyltransferase reaction. Isofagomine and noeuromycin interact with UDP by forming an ion pair interaction between the cationic nitrogen and the β-phosphate of UDP. Isofagomine and noeuromycin also prevented TcdA and TcdB induced cytotoxicity of mammalian cells by preventing glycosylation of Rho GTPases. Finally, in initial proof of concept studies using a mouse model of C. difficile infection, treatment of mice with isofagomine tartrate show potential for protection against C. difficile infection. Isofagomine and noeuromycin represent first-generation analogues that show potential for use as therapeutics against C. difficile pathology and prevention of C. difficile infection.