We conducted a double-blind, vehicle-controlled, dose escalation safety and immunogenicity trial of a candidate herpes simplex virus type 2 (HSV-2) surface glycoprotein D2 (gD2) DNA vaccine administered by use of a needle-free device. Sixty-two healthy adults were randomized using a 4:1 vaccine-to-placebo ratio. Half of the participants were HSV-1 seronegative, and all were HSV-2 seronegative. Vaccine doses included 100 μg, 300 μg, 1,000 μg or 3,000 μg of a plasmid expressing the gD2 protein. Subjects received vaccine at 0, 4, 8, and 24 weeks. Some subjects received an additional 1,000-μg boost at 52 weeks. We found that the vaccine was safe and well tolerated, with most adverse events being local site reactions. No dose-limiting toxicities were observed. gD2-specific cytotoxic T-lymphocyte and lymphoproliferation responses were detected 2 weeks after the third vaccine injection in one of four HSV-1-seronegative, HSV-2-seronegative participants who received 3,000 μg of vaccine. A DNA-based vaccination strategy against HSV-2 appears to be safe and may generate a vaccine-specific cellular immune response, but high vaccine doses are likely needed to elicit an immune response in most vaccinees.