The E protein of Japanese encephalitis virus (JEV) is the major antigen used to elicit neutralizing antibody response and protective immunity in hosts. In this study, the domain III protein of the attenuated strain CH2195LA was cloned to the pET32a expression vector and expressed as a thioredoxin (Trx) fusion protein in Escherichia coli. The recombinant protein was unique in forming a large fraction of the soluble recombinant protein in E. coli. The purified domain III fusion protein (TrxD3) was emulsified in Freund's adjuvant (FA) as well as in different charged liposomes for immunization in mice. Immunization of TrxD3 fusion protein emulsified in Freund's adjuvant and only the cationic liposome resulted in eliciting neutralizing antibodies and protective immunity in ICR mice. The cationic liposome can serve not only as a safer but also an effective adjuvant for the TrxD3 protein immunization. These studies can provide useful information for further developing the domain III recombinant protein vaccine against JEV.