The study was to express prME protein of Japanese encephalitis virus (JEV) in Pichia pastoris and then to evaluate the immunological properties of the recombinant protein in mice, so as to explore a new way for subunit vaccine development of JEV. The JEV prME gene was amplified by RT-PCR with genome RNA of JEV vaccine strain SA14-14-2 and subcloned into pPICZa-A vector, designated as pPICZα-prME. pPICZα-SprME was constructed same as pPICZα-prME besides with the additional 19 Aa signal peptides coding gene of the JEV cap protein C terminal. The linearized expression vector was integrated into the genome of Pichia pastoris X33 under the control of the alcohol oxidase (AOX1) promoter and induced with methanol during fermentation expression. The expression of JEV prME protein was identified by SDS-PAGE and Western blotting, and then it was purified by S-400 High Resolution HiPrep 16/60 Sephacry. The expressed products of Pichia pastoris were visualized by electron microscopy. In the immunization test, four groups of four-week old female mice were immunized subcutaneously with different doses purified JEV prME protein with complete Freund’s adjuvant at a volumetric ratio of 1:1 and a control group was injected with sterile PBS. 10 μg/dose purified JEV prME protein mixing different doses nucleic acid adjuvant (Naa) was vaccinated in mice as the same mode. SDS-PAGE and Western blotting indicate that JEV prME was not cleaved between prM and E during secreted expression in Pichia pastoris. The purified recombinant prME was eluted in the first eluting peak which indicated that its molecular weight about 1×106 Da to 20×106 Da and may form a multimeric. Both the culture supernatant and the purified protein, examined by electron microscopy, we found to contain JEV virus like particles (VLPs) with diameters of 30–50 nm. The anti-JEV VLPs antibody titration reached peak at 3 wpi and still maintained in mice at 7 wpi inoculated with 10 μg and 15 μg prME. The strong antibody response was observed when the mice immunized with prME mixing nucleic acid adjuvant, which elicited high neutralizing antibody titer among 1:80 to 1:160. In conclusion, although JEV prME protein expressed in Pichia pastoris was not cleaved, which formed VLPs and showed efficient immunological properties in mice experiments.