TMF/ARA160 is a Golgi-associated protein with several cellular functions, among them direction of the NF-κB subunit, p65 RelA, to ubiquitination and proteasomal degradation in stressed cells. We sought to investigate the role of TMF/ ARA160 under imposed stress conditions in vivo. TMF-/- and wild-type (WT) mice were treated with the ulcerative agent dextran sulfate sodium (DSS), and the severity of the inflicted acute colitis was determined. TMF-/- mice were found to be significantly less susceptible to DSS-induced colitis, with profoundly less bacterial penetration into the colonic epithelia. Surprisingly, unlike in WT mice, no bacterial colonies were visualized in colons of healthy untreated TMF-/- mice, indicating the constitutive resistance of TMF-/- colonic mucus to bacterial retention and penetration. Gene expression analysis of colon tissues from unchallenged TMF-/- mice revealed 5-fold elevated transcription of the muc2 gene, which encodes the major component of the colonic mucus gel, the MUC2 mucin. Accordingly, the morphology of the colonic mucus in TMF -/- mice was found to differ from the mucus structure in WT colons. The NF-κB subunit, p65, a well known transcription inducer of muc2, was upregulated significantly in TMF-/- intestinal epithelial cells. However, this did not cause spontaneous inflammation or increased colonic crypt cell proliferation. Collectively, our findings demonstrate that absence of TMF/ARA160 renders the colonic mucus refractory to bacterial colonization and the large intestine less susceptible to the onset of colitis.