The host immune response affects pathogen virulence in Clostridium difficile infection (CDI). Thus, cytokine responses to CDI likely are associated with disease initiation and progression. Understanding the molecular drivers of inflammation and biochemical markers of disease severity is important for developing novel therapies and predicting disease prognosis. In this study, we investigated cytokine production in patients with CDI and evaluated the potential of cytokines to serve as biomarkers for CDI and predictors of disease severity. The systemic cytokine profiles of 36 CDI patients (20 with severe disease) and 8 healthy donors and the toxin-induced cytokine profiles of peripheral blood mononuclear cells (PBMC) were determined. Further, we evaluated glucosyltransferase (GT) activity in regulation of toxin-induced cytokine expression. We found upregulation of the majority of measured cytokines (11/20, 55%) in CDI patients. Interleukin-1β (IL-1β), IL-6, IL-8, IL-17A, and IL-16 were the most upregulated. High serum levels of IL-2 and IL-15 were associated with a poor prognosis in CDI patients, whereas high levels of IL-5 and gamma interferon (IFN-γ) were associated with less severe disease. Both TcdA and TcdB were potent inducers of cytokine responses, as demonstrated by stimulation of a greater number and amount of cytokines. In addition to confirming prior reports on the role of IL-8, IL-1β, and IL-6 in CDI, our data suggest that IL-16 and IL-17A, as well as the IL-1β/Th17 axis, play a key role in driving inflammatory responses in CDI. A functional GT domain of C. difficile toxins was required for the induction of a majority of cytokines investigated.