Clostridium difficile is an important nosocomial pathogen of humans. Currently the role of toxin A and toxin B in disease pathogenesis and in the development of severe disease during C. difficile infection (CDI) is poorly characterised. Furthermore, the factors that predispose some patientsto develop mild disease while others develop life-threatening disease are unknown. Understanding how C. difficile modulates the host immune response during infection may provide insights into this phenomenon and may provide a better understanding of the role that each toxin plays in disease. Here, we examined the role of both toxins in pathogenesis and the induction of the host immune response during disease progression. The analysis of isogenic, independently-derived toxin gene mutants in a BI/NAP1/027 C. difficile strain using the mouse model of CDI showed that toxin B plays a critical role in the development of severe disease and in the induction of the host immune response. Increased levels of pro-inflammatory cytokines and chemokines such as C5a, TNF-α, MCP-1 and CXCL1, 9 and 10 were found in colonic tissue isolated from mice infected with a toxin A mutant, at levels similar to those detected in tissues isolated from mice infected with the wild-type strain. Cytokine induction coincided with the development of severe colonic damage and fulminant disease. By contrast, infection with toxin B mutants resulted in only self-limiting diarrhoea and mild disease and led to the induction of significantly lower levels of pro-inflammatory cytokines compared to mice that had been infected with the wild-type strain. These results support the hypothesis that toxin B appears to be the major virulence factor of C. difficile with toxin A playing a more minor role. This study provides valuable insights into the role of toxin A and toxin B in C. difficile disease and the host immune response during infection.