The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is a claudin-4 binder. Very recently, we found that nasal immunization of mice with C-CPE-fused antigen activated antigen-specific humoral and mucosal immune responses and that the deletion of the claudin-4-binding domain attenuated the immune responses. C-CPE-fusion strategy may be useful for mucosal vaccination. C-CPE is a fragment of enterotoxin, and the safety of C-CPE-fused protein is very important for its future application. In the present study, we investigated whether C-CPE-fused antigen induces immune responses without mucosal injury by using ovalbumin (OVA) as a model antigen. Immunohistochemical analysis showed that claudin-4 was expressed in epithelial cell sheets bordering the nasal cavity. Nasal immunization with C-CPE-fused OVA dose-dependently elevated the OVA-specific serum IgG titer, which was 1000-fold greater than the titer achieved by immunization with OVA or a mixture of OVA and C-CPE at 5 microg of OVA. Nasal immunization with C-CPE-fused OVA (5 microg of OVA) activated Th1 and Th2 responses. Histological analysis showed no mucosal injury in the nasal cavity or nasal passage. C-CPE-fused OVA exhibited mucosal vaccination without mucosal injury. These findings indicate thatclaudin-4-targeting using C-CPE can be a potent strategy for mucosal vaccination.