Our gut microbiota plays a protective role in the GI tract, by promoting colonization resistance against enteric pathogens through nutrient competition. Antibiotic treatments in early life can lead to prolonged microbial dysbiosis and increase susceptibility to future enteric infections. Moreover, the gut microbiota have been shown to impact the development of the mucosal and systemic immune systems. Intestinal epithelial cells (IEC) are crucial cells that control interactions between the mucosal immune system and the luminal microbiota, but they also initiate innate immune responses to pathogens. Recent studies have explored the role played by the microbiota in controlling the virulence strategies of the attaching and effacing (A/E) microbe, Citrobacter rodentium (Cr). This pathogen directly adheres to IEC, causing diarrheal disease as well as inflammation, with commensal microbes impacting its ability to infect IEC, as well as being critical for its clearance. At present however, it is not clear whether commensal microbes influence the innate immune response by IEC to Cr infection. Defining the impact of commensal microbes on innate intestinal defenses may prove useful in developing new therapeutic approaches to combat enteric infections. To investigate the impact of commensal microbiota on innate IEC responses to Cr infection. This study compared the innate responses of specific-pathogen free (SPF) and germfree (GF) C57BL/6 mice to Cr infection. Pathogen burdens, pathology scores, qPCR and immunofluorescence staining were used to characterize the mucosal response. SPF and GF mice were orally infected and pathogen burdens determined on day 6 post-infection (6 DPI). Increased pathogen burdens were observed in GF mice as compared to SPF mice. Cecal tissues of GF mice showed increased pathology scores with evidence of increased Cr adherence to the mucosal surface. In contrast, GF mice showed reduced colon pathology scores than SPF mice, with reduced pathogen adherence. Apoptosis was characterized, with the ceca of GF mice at 6 DPI displaying massive IEC death and sloughing, while this was not seen in infected SPF mice. Interestingly, while SPF mice showed the expected upregulation in inflammatory (IL-22, IL-17A, IL-6) and antimicrobial (Reg3γ and Relmβ) responses in both the cecum and colon, GF mice showed a differential response, with dramatically higher levels of IL-22, but reduced expression of Reg3γ and Relmβ as compared to SPF mice. These findings indicate that commensal microbes play a key role in controlling epithelial responses to A/E pathogens, promoting some responses while suppressing others. Further studies will unravel the mechanisms underlying microbiota-based regulation of IEC responses to infection, with the goal of developing novel approaches to prevent human infection. CAG, CIHRFRQS, MSFHR