Both humoral and cell-mediated immune responses are important to protect animals from initial acute viral infection and establishment of chronic infection. Adjuvants for DNA vaccines can influence the balance between humoral and cell-mediated immunities. In this study, a DNA vaccine encoding the hemagglutinin-neuraminidase and fusion genes of Newcastle disease virus (NDV) incorporated with chicken interferon(provax-chIFN-gamma) cDNA as a molecular adjuvant and levamisole (LMS) as a chemical adjuvant was tested for its efficacy in protection against NDV lethal challenge. Compared with DNA vaccine alone, the DNA vaccine with provax-chIFN-gamma plus LMS induced significantly higher humoral and cell-mediated responses, as shown by higher levels of hemagglutination inhibition (HI) titers and T cell proliferation. In addition, the DNA vaccine with provax-chIFN-gamma plus LMS formulation increased the expression of IFN-gamma, interleukin (IL)-2, IL-4, IL-12, and IL-13, suggesting that the effectiveness of the IFN-gamma and LMS formulation is partly due to the enhancement of balanced cytokine production. Furthermore, the two adjuvants yielded 80% protection in chickens against challenge with a lethal dose of the virulent NDV strain. This study demonstrates that the synergistic effects of provax-chIFN-gamma plus LMS as the adjuvants in NDV DNA vaccination could be used to improve protective efficacy in chickens.