Background & Aims: The absence of interleukin 10, a key cytokine in gut homeostasis, causes severe colitis in C3H/HeJBir but not C57BL/6J mice. The major modifier for colitis was mapped on chromosome 3 and designated cytokine deficiency-induced colitis susceptibility 1 (Cdcs1). We developed reciprocal Cdcs1 congenic stocks on both interleukin 10-deficient backgrounds to identify the susceptibility gene and its function. Methods: C3H/HeJBir congenic for the C57BL/6J-derived Cdcs1 allele and reciprocal C57BL/6J congenic for the C3H/HeJBir allele were analyzed for colitis development. Parental strains were compared by electrophoretic mobility shift assay to assess the candidacy of nuclear factor-κB p50 in the Cdcs1 interval. Functional differences were observed in innate and adaptive immune responses of parental and congenic stocks after bacterial ligand exposure in vitro (cytokine release from bone marrow-derived macrophage and dendritic cells) and in vivo (serum cytokines and primed CD4+ T cell proliferation). Results: Cdcs1 was positioned within a minimum 7-megabase interval containing nuclear factor-κB p50. C3H/HeJBir colitis was significantly diminished by the C57BL/6J genome in this interval. Conversely, colitis in C57BL/6J was significantly exacerbated by the reciprocal C3H/HeJBir genome. C3H/HeJBir macrophages constitutively expressed higher nuclear factor-κB p50. Functional assays showed that C3H/HeJBir showed reduced innate responsiveness both in vivo and in vitro to bacterial ligands but showed increased CD4 T-cell responses compared with C57BL/6J. This differential responsiveness was controlled by the respective allele at Cdcs1. Conclusions: The colitogenic Cdcs1 allele impairs innate immunity to bacterial products and in turn skews the adaptive immune response toward compensatory hyperresponsiveness and chronic intestinal inflammation. © 2005 by the American Gastroenterological Association.