Some H5N1 avian influenza viruses (AIVs) are lethal to quail; however, the use of inactivated vaccines in these birds is largely restricted because of side effects caused by oil adjuvants. Here we evaluated the protective efficacy of a DNA vaccine against lethal challenge with H5N1 highly pathogenic avian influenza virus (HPAIV) in quail. Groups of ten 3-wk-old quail were intramuscularly inoculated three times at 3-wk intervals with 10, 15, 30, or 60 µg, respectively, of plasmid pCAGGoptiHA, which expresses a codon-optimized hemagglutinin gene of the H5N1 virus A/goose/Guangdong/1/96 (GS/GD/96). The control group was inoculated with phosphate-buffered saline. Hemagglutination-inhibition (HI) antibodies were monitored every week after the primary vaccination. The quail were challenged intranasally with 10⁵ EID₅₀ of heterologous HPAIV A/duck/Fujian/31/2007 (DK/FJ/31) (H5N1) 2 wk after the third inoculation. Oropharyngeal and cloacal swab specimens were collected 3, 5, and 7 days after inoculation, and quail were observed daily for disease signs and death for 2 wk. The quail showed no side effects after the plasmid inoculation, and HI antibodies were detectable 1wk after the second vaccination in all groups and increased sharply after the third inoculation. All quail in the PBS-inoculated group and 20% of the birds in the 10 µg plasmid-inoculated group died after the lethal H5N1 virus challenge; however, birds in the 15, 30, and 60 µg plasmid-inoculated groups were completely protected. These results indicate that this DNA vaccine holds promise for use in quail to protect against H5N1 AIV.