Commensal bacteria and TLR signaling have been associated with the maintenance of intestinal homeostasis in dextran sodium sulfate-induced intestinal injury. The aim of this study was to determine the in vivo role of TLR/NF-κB activation in a model of commensal bacteria-induced T cell-mediated colitis. A NF-κB reporter gene mouse (NF-κB EGFP)(EGFP, enhanced GFP) was crossed to the colitogenic susceptible strain IL-10−/− and derived into germfree conditions using embryo-transfer technology. Germfree IL-10 wt/wt; NF-κB EGFP and IL-10−/−; NF-κB EGFP mice (wt, wild type) were dual associated with the nonpathogenic commensal bacteria strains Enterococcus faecalis and Escherichia coli. EGFP was detected using macroimaging, confocal microscopy, and flow cytometry. IL-10−/−; MyD88−/− mice were used to assess E. faecalis/E. coli-induced TLR-dependent signaling and IL-23 gene expression. Dual-associated IL-10−/−; NF-κB EGFP mice developed severe inflammation by 7 wk. Macroscopic analysis showed elevated EGFP expression throughout the colon of bacteria-associated IL-10−/−; NF-κB EGFP mice. Confocal microscopy analysis revealed EGFP-positive enterocytes during the early phase of bacterial colonization (1 wk) in both IL-10 wt/wt and IL-10−/− mice, while the signal shifted toward lamina propria T cells, dendritic cells, neutrophils, and macrophages in IL-10−/− mice during colitis (7 wk). The NF-κB inhibitor BAY 11-7085 attenuated E. faecalis/E. coli-induced EGFP expression and development of colitis. Additionally, E. faecalis/E. coli-induced NF-κB signaling and IL-23 gene expression were blocked in bone marrow-derived dendritic cells derived from IL-10−/−; MyD88−/− mice. We conclude that bacteria-induced experimental colitis involves the activation of TLR-induced NF-κB signaling derived mostly from mucosal immune cells. Blocking TLR-induced NF-κB activity may represent an attractive strategy to treat immune-mediated intestinal inflammation.