Nonpathogenic enteric bacterial species initiate and perpetuate experimental colitis in interleukin‐10 geneeficient mice (IL‐10^‐/‐). Bacteria‐specific effects on the epithelium are difficult to distinguish because of the complex nature of the gut microflora. We showed that IL‐10^‐/‐ mice compared to wild‐type mice fail to inhibit pro‐inflammatory gene expression in native intestinal epithelial cells after the colonization with colitogenic Gram‐positive Enterococcus faecalis. Of interest, pro‐inflammatory gene expression was transient after 1 week of E. faecalis monoassociation in IECs from wild‐type mice but persisted after 14 weeks of bacterial colonization in IL‐10^‐/‐ mice. Accordingly, wild‐type IECs expressed phosphorylated NF‐kappaB 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 IECs at days 3, 7, 14, and 28. Consistent with the induction of TLR2‐mediated RelA phosphorylation and pro‐inflammatory 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^‐/‐ IECs. Of note, TGF‐beta‐activated Smad signaling was associated with the loss of TLR2 protein expression and the inhibition of NF‐kappa Bependent gene expression in E. faecalis‐stimulated IEC lines. In conclusion, E. faecalis‐monoassociated IL‐10^‐/‐ but not wild‐type mice lack protective TGF‐beta/Smad signaling and fail to inhibit TLR2‐mediated pro‐inflammatory gene expression in the intestinal epithelium, suggesting a critical role for IL‐10 and TGF‐beta in maintaining normal epithelial cell homeostasis in the interplay with commensal enteric bacteria.