Clostridium difficile is a nosocomial pathogen possessing Surface Layer Proteins (SLPs), which exhibit adherent and immunostimulatory properties. Using bioinformatics tools, evidence of positive selection (PS) was detected in the SLPs of hypervirulent ribotype (RT) 027, indicating a potential correlation between PS and immunostimulatory properties. The SLPs were purified, and examined for their effect on immune cells. Up-regulation of pro-inflammatory cytokines was observed, however the strength of the response differed between strains. RT 027 induced a strong inflammatory response relative to the RT 001, with which it exhibits sequence similarity. An animal model of C. difficile infection compared severity of RT 001 and RT 027. RT 001 induced mild disease with full recovery by day 7, while RT 027 exhibited more severe disease with less clearance. Tissue damage was visualised by histology staining. Further staining showed macrophage and neutrophil infiltration of the tissue. qPCR analysis showed induction of Th17-linked pro-inflammatory cytokines at day 3 in response to RT 001, with levels lowering by day 7. RT 027 infected animals showed later production of IL-10. This may contribute to suppressing clearance. A dendritic cell T cell co-culture was also carried out to fully determine the effect of SLPs on the adaptive immune response. These results strongly suggest the importance of SLPs in disease, and that positive selection may be playing a role in driving the emergence of hypervirulent strains.