Nonpathogenic enteric bacterial species initiate and perpetuate experimental colitis in IL-10 gene-deficient mice (IL-10−/−). Bacteria-specific effects on the epithelium are difficult to dissect due to the complex nature of the gut microflora. We showed that IL-10−/− mice compared with wild-type mice fail to inhibit proinflammatory gene expression in native intestinal epithelial cells (IEC) after the colonization with colitogenic Gram-positive Enterococcus faecalis. Interestingly, proinflammatory gene expression was transient after 1 wk of E. faecalis monoassociation in IEC from wild-type mice, but persisted after 14 wk of bacterial colonization in IL-10−/− mice. Accordingly, wild-type IEC expressed phosphorylated NF-κB subunit RelA (p65) and phosphorylated Smad2 only at day 7 after bacterial colonization, whereas E. faecalis-monoassociated IL-10−/− mice triggered persistent RelA, but no Smad2 phosphorylation in IEC at days 3, 7, 14, and 28. Consistent with the induction of TLR2-mediated RelA phosphorylation and proinflammatory gene expression in E. faecalis-stimulated cell lines, TLR2 protein expression was absent after day 7 from E. faecalis-monoassociated wild-type mice, but persisted in IL-10−/− IEC. Of note, TGF-β1-activated Smad signaling was associated with the loss of TLR2 protein expression and the inhibition of NF-κB-dependent gene expression in IEC lines. In conclusion, E. faecalis-monoassociated IL-10−/−, but not wild-type mice lack protective TGF-β/Smad signaling and fail to inhibit TLR2-mediated proinflammatory gene expression in the intestinal epithelium, suggesting a critical role for IL-10 and TGF-β in maintaining normal epithelial cell homeostasis in the interplay with commensal enteric bacteria.