The envelope (E) protein of Japanese encephalitis virus (JEV) contains 500 amino acids with six "conserved" disulfide bonds to maintain its conformational structure. Neutralizing epitopes located on the E protein are mostly conformational dependent. In this study, we used phage-displayed 12-residue combinatorial peptide libraries to select high-affinity peptide ligands bound to monoclonal antibody E3.3. The specific peptide ligands presented on ten high-affinity phage clones displayed six different amino acid sequences, all showing a novel cis-proline turn structure. After being superimposed onto the best fit of the three-dimensional structure of JEV E protein, these peptide structures were mapped to a conformational region constituted by three continuous polypeptide segments (E307-E309, E327-E333, E386-E390) in domain III. Synthetic peptide ligands based on one peptide sequence (E18) were further investigated using alanine scanning within the cis-proline turn structure to demonstrate its unique molecular characteristics. Our results showed that three residues forming the novel cis-proline turn structure were all important in eliciting JEV-specific neutralizing antibodies in mice.