To explore whether platelet-activating factor (PAF) can disrupt the intestinal epithelial barrier directly and is associated with structural alterations of the F-actin-based cytoskeleton, and to observe the protective effect of intestinal trefoil factor (ITF), we establish an intestinal epithelia barrier model using Caco-2 cells in vitro. Transepithelial electrical resistance and unidirectional flux of lucifer yellow were measured to evaluate barrier permeability; immunofluorescent staining and flow cytometry were applied to observe morphological alterations and to quantify proteins of the F-actin cytoskeleton: the tight junction marker ZO-1 and Claudin-1 were observed using immunofluorescent staining. PAF significantly increased paracellular permeability, at the same time, F-actin and tight junction proteins were disrupted. It was thought that ITF could reverse the high permeability by restoring normal F-actin, ZO-1 and Claudin-1 structures. These results collectively demonstrated that PAF plays an important role in the regulation of mucosal permeability and the effects of PAF are correlated with structural alterations of the F-actin-based cytoskeleton and of tight junctions. ITF can protect intestinal epithelium against PAF-induced disruption by restricting the rearrangement of the F-actin cytoskeleton and of tight junctions.