In this study, we evaluated the antimicrobial properties of XN and C. butyricum culture supernatant (CBs) against C. difficile, focusing on their ability to reduce C. difficile adhesion, inhibit growth, and restrict toxin production. Using a dextran sulfate sodium (DSS)-induced Caco-2 intestinal epithelial model, we demonstrated that XN alleviates C. difficile-induced epithelial barrier disruption and inhibits the activation of related inflammatory signaling pathways. Additionally, our research indicated that both XN and C. butyricum effectively prevent CDI-induced cellular damage and led to significant proteomic regulation in this pathogen. We then mapped the global acetylation landscape of C. difficile proteins response to these two conditions, revealing that lysine acetylation regulates pathogenicity by modulating glycolysis pathways and the activity of metabolic enzymes. These findings suggest that XN and CBs may represent promising therapeutic options for CDI, which serving as a valuable resource for investigating lysine acetylation in C. difficile metabolism and virulence.