Ileal Crohn's disease (CD), a chronic mucosal inflammation, is characterized by two pertinent features: a specific decrease of Paneth cell-produced antimicrobial α-defensins and the presence of mucosal-adherent bacteria. A mutation in NOD2, the muramyl dipeptide recognition receptor, is found in some patients, which leads to an even more pronounced α-defensin decrease. However, the underlying mechanism remains unclear for the majority of patients. In this study, we report a reduced expression in ileal CD of the Wnt-signaling pathway transcription factor Tcf-4, a known regulator of Paneth cell differentiation and α-defensin expression. Within specimens, the levels of Tcf-4 mRNA showed a high degree of correlation with both HD5 and HD6 mRNA. The levels of Tcf-4 mRNA were decreased in patients with ileal disease irrespective of degree of inflammation, but were not decreased in colonic CD or ulcerative colitis. As a functional indicator of Tcf-4 protein, quantitative binding analysis with nuclear extracts from small intestine biopsies to a Tcf-4 high-affinity binding site in the HD-5 and HD-6 promoters showed significantly reduced activity in ileal CD. Furthermore, a causal link was shown in a murine Tcf-4 knockout model, where the comparably reduced expression of Tcf-4 in heterozygous (+/-) mice was sufficient to cause a significant decrease of both Paneth cell α-defensin levels and bacterial killing activity. Finally, the association between Paneth cell α-defensins and Tcf-4 was found to be independent of the NOD2 genotype. This new link established between a human inflammatory bowel disease and the Wnt pathway/Tcf-4 provides a novel mechanism for pathogenesis in patients with ileal CD.