BACKGROUND & AIMS: Immunological disorders of the gastrointestinal tract such as inflammatory bowel disease often result in recurrent and persistently elevated levels of proinflammatory cytokines. Kinase suppressor of Ras 1 (KSR1) is involved in tumor necrosis factor-mediated colon epithelial cell survival, yet its role in chronic inflammation has not been defined. In this study, we tested the hypothesis that KSR1 is protective against spontaneous experimental colitis. METHODS: KSR1(-/-)Interleukin-10 (Il10)(-/-) mice were generated and histolopathologic parameters of intestinal inflammation were scored. Bone marrow transplants performed on wild-type and KSR1(-/-)Il10(-/-) mice determined the contribution of KSR1 in hematopoietic lineages. Mucosal T helper (Th) 1 and Th17 cytokine were also examined. In vitro Th1 and Th17 polarization assays were conducted and interleukin (IL)-17A and interferon-γ (IFN-γ) production analyzed by flow cytometry. Neutralizing antibodies against IgG, IL-17A, or IFN-γ were administered to 3-week-old KSR1(-/-)Il10(-/-) mice for 3 weeks and scored for colitis. RESULTS: KSR1(-/-)Il10(-/-) mice developed accelerated and severe spontaneous colitis by 4 weeks of age. KSR1 expression in hematopoietic lineages was protective against colitis. Both IFN-γ and IL-17A transcripts were elevated in colons of KSR1(-/-) and KSR1(-/-)Il10(-/-) mice. IFN-γ production was increased in lamina propria T cells isolated from KSR1(-/-) and KSR1(-/-)Il10(-/-) mice. Additionally, in vitro Th1 polarization was increased while Th17 polarization was impaired in KSR1-deficient naïve T cells. Finally, administration of IFN-γ neutralizing antibodies attenuated colitis in KSR1(-/-)Il10(-/-) mice. CONCLUSIONS: Mice lacking both KSR1 and IL-10 develop exacerbated colitis due to dysregulated IFN-γ production in T lymphocytes.