Japanese encephalitis virus (JEV) is a flavivirus with a complex life cycle involving mosquito vectors that mainly target birds and pigs, and causes severe encephalitis in children in Asia. Neurotropic flaviviruses of the JEV serogroup have a particular characteristic of expressing a unique nonstructural NS1' protein, which is a prolongation of NS1 at the C terminus by 52 amino acids derived from a pseudoknot-driven-1 translation frameshift. Protein NS1' is associated with virus neuroinvasiveness. In this study, the need of the pseudoknot structure for NS1' synthesis was confirmed. By using a specific antibody against the prolonged peptide, NS1' was found to be absent from the JEV SA14-14-2 vaccine strain, resulting from a single nucleotide silent mutation in the pseudoknot. A partial cleavage of NS1' at a specific site of its C-terminal appendix recognized by caspases and inhibited by caspase inhibitors suggests a unique feature of intracellular NS1'.