Hypervirulent strains of Clostridium difficile have emerged over the past decade, increasing the morbidity and mortality of patients infected by this opportunistic pathogen. Recent work suggested the major C. difficile virulence factor, TcdB, from hypervirulent strains (TcdB(HV)) was more cytotoxic in vitro than TcdB from historical strains (TcdB(HIST)). The current study investigated the in vivo impact of altered TcdB tropism, and the underlying mechanism responsible for the differences in activity between the two forms of this toxin. A combination of protein sequence analyses, in vivo studies using a Danio rerio model system, and cell entry combined with fluorescence assays were used to define the critical differences between TcdB(HV) and TcdB(HIST). Sequence analysis found that TcdB was the most variable protein expressed from the pathogenicity locus of C. difficile. In line with these sequence differences, the in vivo effects of TcdB(HV) were found to be substantially broader and more pronounced than those caused by TcdB(HIST). The increased toxicity of TcdB(HV) was related to the toxin's ability to enter cells more rapidly and at an earlier stage in endocytosis than TcdB(HIST). The underlying biochemical mechanism for more rapid cell entry was identified in experiments demonstrating that TcdB(HV) undergoes acid-induced conformational changes at a pH much higher than that of TcdB(HIST). Such pH-related conformational changes are known to be the inciting step in membrane insertion and translocation for TcdB. These data provide insight into a critical change in TcdB activity that contributes to the emerging hypervirulence of C. difficile.