Fibronectin (FN) is a multifunctional extracellular matrix protein that plays an important role in cell proliferation, adhesion, and migration. FN expression or its role in colitis is not known. The goal of this study is to characterize FN expression, regulation, and role during intestinal inflammation. Wild-type and transgenic mice expressing luciferase under the control of the human FN promoter, given water or 3% dextran sodium sulfate, were used as animal models of colitis. The Caco2-BBE model intestinal epithelial cell line was used for in vitro studies. FN protein is abundantly expressed by surface epithelial cells in the normal colon. Immunohistochemistry and luciferase assay in mice expressing the FN promoter linked to luciferase demonstrated that FN synthesis was up-regulated during colitis, during both the acute phase and the healing phase. In vitro experiments demonstrated that FN increased the expression of the FN integrin receptor alpha5beta1 in a dose- and time-dependent manner. FN also induced the expression and activation of NF-kappaB. Further, FN potentiated Caco2-BBE cell attachment and wound healing, which was inhibited by RGD peptide as well as NF-kappaB inhibitors MG-132 and 1-pyrrolidinecarbodithioic acid, ammonium salt. In conclusion, FN is abundantly expressed and synthesized by colonic epithelial cells. FN is transcriptionally up-regulated in epithelial cells during both the dextran sodium sulfate-induced colitic and the recovery phase. FN enhances cell attachment and wound healing, which is dependent on binding to the integrin receptor and the NF-kappaB signaling. Together our data show that epithelial-derived FN potentiates cell attachment and wound healing through epithelial-matrix interactions and that FN expression may have important implications for maintaining normal epithelial integrity as well as regulating epithelial response to injury during colitis.