Wnt signaling is essential for the maintenance of adult stem cells and its aberrant activation is a stimulator of carcinogenesis. The transmembrane protein, Wntless, is an essential Wnt signaling component through regulating the secretion of Wnt ligands. Here, we generated a mouse model with specific Wntless knockout in intestinal epithelium to study its function in the intestinal epithelium. Wntless knockout exhibits no obvious defects in mice but significantly disrupted proliferation and differentiation of small intestinal organoids. We also discovered that these deficiencies could be partially rescued by Wnt3a supplement but not Wnt9b. To further investigate the role of Wntless in tumorigenesis, APC-deficient spontaneous intestinal tumors and chemical induced colorectal cancer mouse models were employed. To our surprise, intestinal epithelium-specific knockout of Wntless did not cause significant differences in tumor number and size. In summary, our data demonstrated that epithelial Wntless was required for the growth and differentiation of small intestinal organoids but not in live animals, suggesting the other tissues, such as mesenchymal tissue, play critical role for Wnt secretion in both intestinal homeostasis as well as tumorigenesis. (C) 2019 Elsevier Inc. All rights reserved.