On the basis of previous studies demonstrating that a breach of the colonic epithelial barrier is associated with a microbiota-dependent increase in lamina propria (LP) regulatory cells, we investigated if the lack of spontaneous intestinal inflammation observed in nucleotide-binding oligomerization domain 2 (Nod2)-/- mice was due to enhanced intestinal regulatory function. We found that the LP CD4+ T-cell population of Nod2-/- mice contains an increased percentage of CD4+ regulatory T cells bearing transforming growth factor -β/latency peptide (LP CD4+LAP (latency-associated peptide) + T cells) both under baseline conditions and following an intentional breach of the colonic barrier induced by ethanol administration. In addition, we found that Nod2-/- mice manifest decreased severity of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-colitis and that TNBS-colitis in Nod2-/- or Nod2+/+ mice is ameliorated by adoptive transfer of LP cells from ethanol-treated mice before, but not after, depletion of LAP+ T cells. This increased regulatory T-cell response in Nod2-/- mice could explain why NOD2 polymorphisms in humans are not in themselves sufficient to establish inflammatory lesions.