Clostridium difficile is an important nosocomial pathogen of humans and the leading cause of infectious diarrhoea in hospitals worldwide. C. difficile infection (CDI) is induced by antibiotic treatment or disruption of the normal gastrointestinal microbiota. Infection with toxigenic C. difficile strains causes extensive colonic inflammation, epithelial tissue damage and rapid fluid loss, which manifests as diarrhoea. C. difficile damages the colonic epithelium via the action of two toxins, TcdA and TcdB. Although a better understanding of the host immune response to C. difficile infection has been gained over recent years, the effect of infection on stem cells and gut regeneration is unknown. The role that each of the major toxins plays in these host effects is also not understood. Using a mouse model, we show that intestinal integrity and regeneration is altered by C. difficile infection, with a disruption in colonic adherens-junctions and crypt polarity seen, as well as an alteration in stem cell function. These effects are primarily mediated by TcdB. TcdB also targets human colonic organoids, suggesting that gut regeneration during human C. difficile infection may be diminished. These results enhance our current understanding of C. difficile pathogenesis and identify new targets for therapeutics that aid gut repair following toxin-mediated damage, which is particularly relevant for recurrent C. difficile infection.