Clostridium difficile is an important nosocomial pathogen of humans and the leading cause of infectious diarrhoea in hospitals worldwide. C. difficile infection 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 colonic stem cells and gut epithelial regeneration is unknown. The role that each of the major toxins plays in mediating effects on the host tissue is also not understood. To address this we used a mouse model of C. difficile infection and analysed an isogenic panel of independently derived toxin gene mutants in an epidemic C. difficile strain, over a defined time course of infection. Our studies show that intestinal integrity is altered by C. difficile infection, with a disruption in colonic adherens-junctions and epithelial cell polarity observed. Using in vitro colonic organoids as a method to analyse stem function in tissue from infected animals we have identified key alterations in stem cell function in a time dependent manner. These effects are primarily mediated by TcdB. TcdB, but not TcdA, also targets human colonic organoids, suggesting that epithelial renewal following human C. difficile infection may be diminished. These results enhance our current understanding of C. difficile pathogenesis with the potential to identify new targets for therapeutics that aid gut repair following toxin-mediated damage, which is particularly relevant for recurrent C. difficile infection.