Background: The intestinal epithelium accommodates with a myriad of commensals to maintain immunological homeostasis, but the underlying mechanisms regulating epithelial responsiveness to flora-derived signals remain poorly understood. Herein, we sought to determine the role of the Toll/interleukin (IL)-1 receptor regulator Toll-interacting protein (Tollip) in intestinal homeostasis. Methods: Colitis susceptibility was determined after oral dextran sulfate sodium (DSS) administration or by breeding Tollip(-/-) on an IL-10(-/-) background. The intestinal flora was depleted with 4 antibiotics before DSS exposure to assess its contribution in colitis onset. Bone marrow chimeras were generated to identify the cellular compartment, whereby Tollip may negatively regulate intestinal inflammation in response to DSS. Tollip-dependent epithelial barrier functions were studied in vitro by using Tollip-knockdown in Caco-2 cells and in vivo by immunohistochemistry and fluorescein isothiocyanate-labeled dextran gavage. Results: Genetic ablation of Tollip did not lead to spontaneous intestinal inflammatory disorders. However, Tollip deficiency aggravated spontaneous disease onset in IL-10(-/-) mice and increased susceptibility to DSS colitis. Increased colitis severity in Tollip-deficient mice was not improved by bacterial flora depletion using broad-spectrum antibiotics. In addition, DSS exposure of bone marrow chimeric mice revealed a protective role for Tollip in nonhematopoietic cells. Knockdown of Tollip in epithelial cells led to exaggerated NF-B activity and proinflammatory cytokine secretion. Finally, DSS-treated Tollip(-/-) mice showed enhanced intestinal permeability and increased epithelial apoptosis when compared with wild-type controls, a finding that coincided with tight junction alterations on injury. Conclusion: Overall, our data show an essential role for Tollip on colitis susceptibility in mice.