Transcription factor NF-kappa B has both pro-apoptotic and anti-apoptotic properties depending on the cell type. Its role in the intestinal epithelial cell has not been well elucidated. Trefoil factor 3 (TFF3) is an anti-apoptotic peptide secreted by intestinal goblet cells. Here we show that TFF3 activated NF-kappa B p50/p65 heterodimer within 1 h in IEC-18 cells (a nontransformed rat intestinal epithelial cell line). Moreover, we found that TFF3-treated IEC-18 cells are resistant to anoikis, an anchorage-related apoptosis in epithelium. In addition, the stable expression of a mutant form of the endogenous NF-kappa B inhibitor (I kappa B alpha(mut)) in IEC-18 cells results in a significant attenuation of anti-anoikic effect of TFF3. Taken together, these data indicate that (1) TFFS is an endogenous gastrointestinal peptide with anti-anoikic property; (2) TFF3 activates NF-kappa B in enterocytes; and (3) TFF3-induced resistance to anoikis in intestinal epithelial cells is mediated by a distinct signaling cascade linked to NF-kappa B. Furthermore, our study implicates NF-kappa B as an important regulator in survival pathway of intestinal epithelial cells,