The current treatments available for C. difficile infections (CDI) aims to eliminate C. difficile bacteria, however they do not treat the inflammation and tissue damage triggered by the virulence factors, TcdA and TcdB. Indeed, enhanced chemokine/cytokine expression during CDI is associated with poor clinical outcomes. The xenobiotic receptor, pregnane X receptor (PXR) can be activated by antibiotics which are known to target C. difficile. The PXR has also been reported to attenuate intestinal inflammation by inhibiting NFκB signaling. Therefore we hypothesized that targeting the PXR would be an effective strategy to reduce C. difficile toxin-induced inflammation and tissue damage. Activation of the PXR attenuated C. difficile toxin-induced inflammatory mediator release in Caco-2 intestinal epithelial cells (IECs) and in mouse enteroid derived colonic monolayers. In vivo studies demonstrated that PXR activation using the pharmacological agonist pregnenolone-16-α-carbonitrile (PCN) attenuated the expression of pro-inflammatory mediators and significantly reduced neutrophil infiltration to the colonic lamina propria following toxin challenge. Additionally, when compared to wild type mice, PXR-/- mice challenged with toxin displayed a hyper-susceptible phenotype with significant eosinophilic inflammation and increased pro-inflammatory gene expression. Contrary to our hypothesis, this phenotype was not mediated by the loss of the PXR in the epithelium, as determined by the use of a PXRfl/fl x villin cre+ knockout mouse. Taken together, these data will help us expand our knowledge of the PXR’s role in host-pathogen interactions, and its potential to be modulated for the treatment of inflammation and tissue damage in CDI.