Folate receptor α (FRα) is overexpressed in a variety of solid tumors and has become an attractive target antigen for immunotherapy purposes. A DNA vaccine was generated by ligation of FRα cDNA into the eukaryotic vector pcDNA3.1. Expression of FRα was confirmed in transiently transfected B16 cells. B16 cell lines that stably express FRα were set up by G418 selection. A total of 100 µg purified plasmid DNA alone or in combination with CpG oligodeoxynucleotides (CpG ODN) was injected intramuscularly in C57BL/6 mice four times at one week intervals. ELISA analysis confirmed that high titers of antibodies against FRα existed in the sera of the experimental animals. Specific cytotoxic T lymphocyte activity against FRα-expressing B16 cells was found and FRα specific lymphocyte proliferation was detected. Coinjection of CpG ODN increased both humoral and cellular immune responses. In the protective model, in which C57BL/6 mice were immunized with the FRα DNA vaccine four weeks before tumor cell inoculation, the growth of tumor was significantly inhibited, and the presence of CpG ODN further increased the inhibitory effect. FRα DNA vaccine alone did not show a significant inhibitory effect in the therapeutic model, in which the DNA vaccine was immediately injected after tumor inoculation. However, FRα DNA vaccine plus CpG ODN showed a significant inhibitory effect in tumor growth. Survival curves for both animal experiments confirmed that mice immunized with pcDNA3.1/FRα plus CpG ODN had a significantly prolonged survival period than that of the pcDNA3.1 control group, the CpG ODN group or the pcDNA3.1/FRα group. The above showed that human FRα based DNA vaccination with CpG ODN as an adjuvant was effective in growth inhibition of a FRα expressing tumor in mice and deserves further evaluation as a possible immunotherapy.