The envelope glycoprotein (E) of flavivirus is the major structural protein on the surface of the mature virions. The complexes of premembrane (prM) and E play important roles in virus assembly and fusion modulation and in potential immunity-inducing vaccines. In the present study, the cDNA encoding prM and E proteins of dengue virus type 2 (DENV-2) was subcloned into the pGAPZalphaA vector and further integrated into the genome of Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. The high-level constitutive expression of recombinant E antigen was achieved in P. pastoris. Both the cell lysate and the culture supernatant, examined by electron microscopy, were found to contain DENV-2 virus-like particles (VLPs) with diameters of about 30 nm. After immunization of BALB/c mice, the VLPs exhibited similar efficacies as inactivated virus in terms of antibody induction and neutralization titer. These results suggest that recombinant DENV VLPs can be efficiently produced in the GAP promoter-based P. pastoris expression system. This system may be useful for the development of effective and economic dengue subunit vaccine.