Abstract: The unpredictable fast evolution of H3N8 equine Influenza virus and its recent transmission to dogs have raised the importance of a rapid vaccine production strategy. Currently available EIV vaccines are problematic due to their production cost, time and transportation restrictions. DNA Plasmids expressing immunogenic gene (s) are good alternative as it has the advantages of modified living vaccines without the risks of infection and/or reversion to virulence as well as ease of production and handling. In this study, DNA plasmid expressing HA1 (PcDNArHA1) of an H3 Egyptian equine influenza isolate was prepared and its immunogenicity in female wistar rats was evaluated. HA1 protein of PcDNArHA1 was expressed in Vero cell line and detected in the cytoplasm by indirect immunofluorescence. Two doses of ultrapurified PcDNArHA1, 100 μg each were then inoculated 15 day apart in the quadriceps muscle of female wistar rats test group while placebo group inoculated only PBS. Rats were euthanized and the serum collect.