Enterotoxigenic Escherichia coli (ETEC) is a major pathogen that evokes acute diarrhea among children worldwide and travelers to developing countries. However, there is no ideal vaccine against it yet. In an effort to develop a subunit vaccine for ETEC, a translational fusion with cholera toxin B subunit (CTB) upstream of CS3 was constructed. The fusion protein synthesized in E.coli had a molecular mass of 29 ku, as expected and retained the antigenicity of both CTB and CS3 as confirmed by Western blot analysis with the polyclonal anti-CTX rabbit serum and the monoclonal anti-CS3 mouse serum, respectively. The 6×His-tagged CTB/CS3 protein was purified by Ni-NTA affinity chromatography followed by renaturation. A fraction of the fusion protein could form pentamers and these pentamers retained the ability to bind GM1-ganglioside. Mice immunized by intraperitoneal injection with the fusion protein produced anti-CTB and anti-CS3 serum IgG and secretory IgA. Furthermore, it was shown that fusion to CTB increased the systemic and mucosal immune responses against CS3 to some extent.