In this study, the immune-modulatory and vaccine effects of using an interleukin (IL)-18 expression plasmid as a genetic adjuvant to enhance DNA vaccine-induced immune responses were investigated in a mouse herpes simplex virus 1 (HSV-1) challenge model. BALB/c mice were immunized by three intramuscular inoculations of HSV-1 glycoprotein D (gD) DNA vaccine alone or in combination with a plasmid expressing mature IL-18 peptide. Both the serum IgG2a/IgGl1 ratio and T helper 1-type (Th1) cytokines [IL-2 and interferon (IFN)-gamma] were increased significantly by the co-injection of the IL-18 plasmid compared with the injection of gD DNA alone. However, the production of IL-10 was inhibited by IL-18 plasmid co-injection. Furthermore, IL-18 plasmid co-injection efficiently enhanced antigen-specific lymphocyte proliferation and the delayed-type hypersensitivity response. When mice were challenged with HSV-1 at the cornea, co-injection of IL-18 plasmid with gD DNA vaccine showed significantly better protection, manifested as lower corneal lesion scores and faster recovery. These experiments indicate that co-injection of an IL-18 plasmid with gD DNA vaccine efficiently induces Th1-dominant immune responses and improves the protective effect against HSV-1 infection.