Clostridium difficile (C. difficile) is a gram-positive, spore-forming, pathogenic bacterium that results in a range of gastrointestinal diseases. The incidence of C. difficile infection (CDI) has increased dramatically and has a significant impact on healthcare settings worldwide. The severity of disease may be dependent on the ribotype (RT) of C. difficile present. Previous research from our laboratory has shown that Surface Layer Proteins (SLPs) from RT 001 activate Toll-like receptor 4 (TLR4), with subsequent activation of downstream signalling pathways known to be important in the clearance of CDI. In this study we demonstrate that SLPs from RT 001 fail to activate IRF3 signalling, while SLPs from RT 027 activate both arms of the TLR4 pathway. Evidence from the literature suggests that microRNAs (miRNAs) tightly regulate TLR4 signalling and have a role in orchestrating the host’s immune response to infection. The profile of miRNAs regulated in response to SLPs from C. difficile has not been profiled before. Subsequently we identified novel miRNAs regulated in response to LPS, SLPs from RT 001 and RT 027 in vitro. We found 24 miRNAs were differentially regulated between SLPs in response to RT 001 and RT 027 and there was a global down regulation of miRNAs in response to SLPs from RT 027. These miRNAs may modulate TLR4 signalling. Data from colonic tissue, from an in vivo murine model show miR-146a, miR-145, miR-155 and let-7e may have a role in regulating the host’s immune response during early and late stage CDI. The absence of miRNAs regulated in response to RT 027 may correlate to less efficient clearance by the host’s immune response and more persistent infection. The miRNAs are predicted to target essential cell processes and the impact of the modulation of the immune response by these miRNAs may lead to biologically relevant changes at the cell level. Further work is needed to fully elucidate the complexities of these miRNAs in relation to the networks they modulate. The effectiveness of current treatments is limited by a lack of response in some patients and high recurrence rates. The data generated in this study may be used to develop miRNA based therapy for the treatment of persistent CDI, allowing bacterial clearance by the host’s immune system without the need for antibiotics.