Highly pathogenic avian influenza viruses (HPAIV) have historically caused disastrous damage to the poultry industry, and recently they have shown their zoonotic potential by causing human infections and deaths. Control and prevention of HPAIV are therefore important issues for both veterinary and human public health. In this study, we constructed a plasmid, pCAGGoptiH7, encoding a codon-optimized HA gene of the H7N1 avian influenza virus A/FPV/Rostock/34 (RK/34). To evaluate the vaccine efficacy of pCAGGoptiH7, groups of specific-pathogen-free (SPF) chickens were intramuscularly inoculated with one or two doses of 100 µg, 50 µg, or 10 µg of the plasmid in 3-wk intervals. Four weeks after the single vaccination or 2 wk after the second dose, all chickens were challenged with 100CLD₅₀ (chicken lethal dose) of highly pathogenic RK/34. After the single dose vaccination, only 90% of chickens were protected in all of the pCAGGoptiH7-immunized groups, although all of the chickens immunized generated detectable HI antibodies. After the second dose of vaccination, HI antibodies increased sharply, and chickens in the 100-µg and 50-µg pCAGGoptiH7-immunized groups were completely protected from virus challenge (no disease signs, no virus shedding, and no deaths). Low titers of virus shedding were detected in two out of ten chickens inoculated with two doses of 10-µg pCAGGoptiH7, although no disease or death was observed. These results provide a strong argument for the continued evaluation of this vaccine in field trials.