Claudin family proteins, which contain 4 transmembrane domains, play a pivotal role in the barrier function of tight junctions (TJs) in epithelial sheets. We previously found that a modulator of claudin-4, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is a potent enhancer of jejunal drug absorption in rats. But the effects of C-CPE on the barrier function of TJs have never been fully understood. In the present study, we investigated the effects of C-CPE on the barrier function of TJs in Caco-2 monolayer and characterized the functional domain of C-CPE that is responsible for interaction with claudin-4. To evaluate the effects of C-CPE on the barrier function of TJs, we measured transepithelial electric resistance (TER) in Caco-2 monolayer cells seeded onto polycarbonate filters. Treatment of Caco-2 cells with C-CPE resulted in a decrease in TER. But, deletion of the 30 C-terminal amino acids of C-CPE, which is the putative binding domain for claudin, attenuated the decrease in TER values. Moreover, ablation of the 16 C-terminal amino acids of C-CPE also resulted in attenuation of the decrease in TER values. The C-terminal-deleted C-CPEs did not interact with claudin-4 or the extracellular domain 2 of claudin-4, which is the C-CPE binding site. These results suggest that the 16 C-terminal amino acids of C-CPE are responsible for the interaction of C-CPE and claudin-4 following the disruption of TJ barrier function.