Finding differences in systemic inflammatory response in ulcerative colitis (UC), UC with Clostridium difficile infection (CDI), and CDI could lead to a better ability to differentiate between UC with symptomatic CDI and UC with C. difficile colonization, and could identify specific inflammatory pathways for UC or CDI, which could be therapeutic targets. We prospectively collected sera from symptomatic UC patients whose stools were tested for toxigenic C. difficile, and from CDI patients who did not have UC (CDI-noUC). The UC patients with positive tests (UC-CDI) were further categorized into responders to CDI treatment (UC-CDI-R) and non-responders (UC-CDI-NR). We compared serum inflammatory mediators among groups using unadjusted and adjusted multivariable statistics. We included 117 UC [27 UC-CDI, 90 UC without CDI (UC-noCDI)] and 16 CDI-noUC patients. Principal component analysis (PCA) did not reveal significant differences either between UC-CDI and UC-noCDI groups, or between UC-CDI-R and UC-CDI-NR groups. In contrast, the PCA showed significant separation between the UC and CDI-noUC groups (P = 0.002). In these two groups, hepatocyte growth factor (HGF) and chemokine (C-C motif) ligand 2 (CCL2) levels were significantly lower and IL-23 levels were higher in UC patients in multivariable analyses. The model to distinguish UC from CDI including IL-23, HGF, CCL2, age, gender, and HGB had an AuROC of 0.93. Inflammatory profiles could not distinguish UC-CDI from UC-noCDI, and UC-CDI-R from UC-CDI-NR. However, the UC and CDI-noUC groups were significantly different. Future work should examine whether therapeutic agents inhibiting IL-23 or stimulating HGF can treat UC.