Transcription factor NF-κB 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-κB 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-κB inhibitor (IκBα^mut) in IEC-18 cells results in a significant attenuation of anti-anoikic effect of TFF3. Taken together, these data indicate that (1) TFF3 is an endogenous gastrointestinal peptide with anti-anoikic property; (2) TFF3 activates NF-κB in enterocytes; and (3) TFF3-induced resistance to anoikis in intestinal epithelial cells is mediated by a distinct signaling cascade linked to NF-κB. Furthermore, our study implicates NF-κB as an important regulator in survival pathway of intestinal epithelial cells.