The use of specific combinations of antigens and adjuvant represents a promising approach for increasing the immunogenicity of DNA vaccines. In the present study, we evaluated the immunity and antitumor effects of DNA vaccines with G250 as the target antigen in a mouse model of renal cell carcinoma. We constructed two recombinant plasmids, pVAX1-G250 and pVAX1-CD40L. The recombinant plasmids were injected into mice by intramuscular injection and electrical pulse stimulation. ELISA and ELISPOT experiments were performed to evaluate the corresponding humoral and cellular immune responses following immunization. To further investigate the antitumor potential of the DNA vaccines, we established a tumor-bearing mouse model expressing G250 target antigen. Our results showed that immunization with the combination of the two plasmids exerted the strongest anti-tumor effects. Therefore, our findings demonstrated the effectiveness of CD40L as an adjuvant for DNA vaccines and highlighted the promising use of these vaccines for the treatment of tumors.