Trefoil factor 3 (intestinal trefoil factor) is a cytoprotective factor in the gut. Here, we compared the effect of trefoil factor 3 with tumor necrosis factor- on (1) activation of nuclear factor-B in intestinal epithelial cells; (2) expression of Twist protein (a molecule essential for down-regulation of nuclear factor-B activity in vivo); and (3) production of interleukine-8. We showed that Twist protein is constitutively expressed in intestinal epithelial cells. Tumor necrosis factor- induced persistent degradation of Twist protein in intestinal epithelial cells via a signal pathway linked to proteasome, which was associated with prolonged activation of nuclear factor-B. In contrast to tumor necrosis factor, trefoil factor 3 triggered transient activation of nuclear factor-B and prolonged up-regulation of Twist protein in intestinal epithelial cells via an ERK kinase-mediated pathway. Unlike tumor necrosis factor-, transient activation of NF-B by trefoil factor 3 is not associated with induction of IL-8 in cells. To examine the role of Twist protein in intestinal epithelial cells, we silenced the Twist expression by siRNA. Our data showed that trefoil factor 3 induced interleukine-8 production after silencing Twist in intestinal epithelial cells. Together, these observations indicated that (1) trefoil factor 3 triggers a diverse signal from tumor necrosis factor- on the activation of nuclear factor-B and its associated molecules in intestinal epithelial cells; and (2) trefoil factor 3-induced Twist protein plays an important role in the modulation of inflammatory cytokine production in intestinal epithelial cells.