Abstract: OBJECTIVE To analyze the influence of Clostridium difficile infection（CDI） on mononuclear cell Toll-like receptor 4（TLR4）/nuclear factor-kappa B（NF-κB）（TLR4/NF-κB） signaling pathways and interleukin-27（IL-27） expression level in patients with ulcerative colitis（UC）. METHODS Total of 157 patients with UC and CDI admitted to Hubei Xiangyang Central Hospital between Jan 2018 and Dec 2019 were recruited and divided into the CDI group and 157 patients with UC and without CDI during the same period were in the non-CDI group. The TLR4/NF-κB signaling pathway indicators and IL-27 levels between the two groups were compared. Stepwise logistic regression analysis was performed to analyze the relationship between CDI and expression of TLR4/NF-κB signaling pathway and IL-27 in patients with UC. RESULTS There were statistically significant differences between the two groups in terms of clinical type, the extent of the disease, activity, activity level, endoscopic activity classification, laboratory indicators, use of proton pump inhibitor（PPI）, use of 5-aminosalicylic acid and use of glucocorticoids（P＜0.05）. The levels of LR4 mNRA, NF-κB mNRA, tumor necrosis factor-α（TNF-α） mRNA, interleukin-6（IL-6） mNRA and interleukin-1β（IL-1β） mNRA in the CDI group were significantly higher than those in the non-CDI group（P＜0.05）. The levels of TLR4 mRNA, NF-κB mNRA, TNF-α mRNA, IL-6 mNRA, IL-1β mNRA and IL-27 showed significant differences between two groups of patients with different disease activity, different activity levels, and different endoscopic grades（P＜0.05）. Logistic regression analysis showed that TLR4 mRNA, NF-κB mNRA, TNF-α mRNA, IL-6 mNRA, IL-1βmNRA, IL-27, UC activity level and hemoglobin were independent influencing factors for CDI in patients with UC. CONCLUSION CDI promotes the overactivation of TLR4/NF-κB signaling pathway and the overexpression of IL-27 in patients with UC, which is closely related to the occurrence of CDI.