Periostin is a unique extracellular matrix protein, deposition of which is enhanced by mechanical stress and the tissue repair process. Its significance in normal and neoplastic colon has not been fully clarified yet. Using immunohistochemistry and immunoelectron microscopy with a highly specific monoclonal antibody, periostin deposition was observed in close proximity to pericryptal fibroblasts of colonic crypts. The pericryptal pattern of periostin deposition was decreased in adenoma and adenocarcinoma, preceding the decrease of the number of pericryptal fibroblasts. Periostin immunoreactivity appeared again at the invasive front of the carcinoma and increased along the appearance of cancer-associated fibroblasts. ISH showed periostin signals in cancer-associated fibroblasts but not in cancer cells. Ki-67–positive epithelial cells were significantly decreased in the colonic crypts of periostin^−/- mice (∼0.6-fold) compared with periostin^+/+ mice. In three-dimensional co-culture within type I collagen gel, both colony size and number of human colon cancer cell line HCT116 cells were significantly larger (∼1.5-fold) when cultured with fibroblasts derived from periostin^+/+ mice or periostin-transfected NIH3T3 cells than with those from periostin^−/- mice or periostin–non-producing NIH3T3 cells, respectively. Periostin is secreted by pericryptal and cancer-associated fibroblasts in the colon, both of which support the growth of epithelial components.