Clostridium difficile is a leading cause of antibiotic-associated diarrhoea, a significant animal pathogen, and a worldwide public health burden. Most disease-causing strains secrete two exotoxins, TcdA and TcdB, which are considered to be the primary virulence factors, with some strains also producing a third toxin known as CDT. Understanding the role that these toxins play in disease is essential for the rational design of urgently needed new therapeutics. Their relative contribution to disease however remains contentious. Using three different animal models, we show that TcdA+TcdB- mutants are attenuated in virulence in comparison to the wild type (TcdA+TcdB+) strain whereas TcdA-TcdB+ mutants are fully virulent. Furthermore, we show that TcdB alone is associated with severe local and systemic disease. Finally, host gene expression analysis following infection with the wild type strain and toxin mutants revealed unique transcriptomic signatures to each strain, suggesting that clinical isolates of C. difficile might elicit a differential host response depending on the particular toxins produced by the infecting strain. Under the conditions tested CDT binary toxin had little impact on disease severity. Importantly, we also found that the animal model used profoundly influenced disease outcomes, which has important ramifications for the validation of new therapeutics and disease pathogenesis studies. Overall, our results show unequivocally that TcdB is the major virulence factor of C. difficile, causing severe gut destruction and mediating damage to remote organs beyond the colonic niche. This work provides new insights into the role that localised gastrointestinal infections play in systemic host damage and highlights the critical nature of using appropriate and, when possible, multiple animal infection models to study bacterial virulence mechanisms.