Granulocyte/macrophage colony-stimulatory factor (GM-CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen-presenting cells (APCs) to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells (DCs). This study evaluated the utility of GM-CSF cDNA as a DNA vaccine adjuvant for glycoprotein B (gB) of pseudorabies virus (PrV) in a murine model. The co-injection of GM-CSF DNA enhanced the levels of serum PrV-specific IgG with a 1.5-to 2-fold increase. Moreover, GM-CSF co-injection inhibited the production of IgG2a isotype. However, it enhanced production of an IgG1 isotype resulting in humoral responses biased to the Th2-type against PrV antigen. In contrast, the co-administration of GM-CSF DNA enhanced the T cell-mediated immunity biased to the Th1-type, as judged by the significantly higher level of cytokine IL-2 and IFN-gamma production but not IL-4. When challenged with a lethal dose of PrV, the GM-CSF co-injection enhanced the resistance against a PrV infection. This suggests that co-inoculation with a vector expressing GM-CSF enhanced the protective immunity against a PrV infection. This immunity was caused by the induction of increased humoral and cellular immunity in response to PrV antigen.