Nucleotide oligomerisation domain 2 (NOD2) mutations are associated with susceptibility to Crohn's disease and graft-versus-host disease, two human disorders related with dysfunctions of Peyer's patches (PPs). In Nod2^−/− mice transcellular permeability and bacterial translocation are increased in PPs. In this study, we show that both anti-CD4⁺ and anti-interferon γ (anti-IFNγ) monoclonal antibodies abrogate this phenotype and reduce the expression of tumour necrosis factor (TNF) receptor 2 and the long isoform of myosin light chain kinase, thus demonstrating that immune T cells influence the epithelial functions. In turn, intraperitoneal injection of ML-7 (a myosin light chain kinase inhibitor) normalises the values of CD4⁺ T cells, IFNγ and TNFα. This reciprocal cross-talk is under the control of the gut microflora as shown by the normalisation of all parameters after antibiotic treatment. Toll-like receptor 2 (TLR2) and TLR4 expression were increased in Nod2^−/− mice under basal conditions and TLR2 and TLR4 agonists induced an increased transcellular permeability in Nod2^+/+ mice. Muramyldipeptide (a Nod2 agonist) or ML-7 was able to reverse this phenomenon. It thus appears that Nod2 modulates the cross-talk between CD4⁺ T cells and the epithelium recovering PP and that it downregulates the pro-inflammatory effect driven by the ileal microflora, likely by inhibiting the TLR pathways.