The 2004 Asian H5N1 epizootic outbreak indicates the urgent need for vaccines against highly pathogenic avian influenza (HPAI) virus. The manufacture of inactivated whole-virus vaccines from HPAI viruses by traditional methods is not feasible for safety reasons as well as technical issues. The low pathogenic avian influenza A/wild bird feces/CSM2/02 (H5N3) virus was used as a heterologous neuraminidase vaccine, and HPAI A/CK/Korea/ES/03 (H5N1) virus was used as a homologous neuraminidase vaccine. Protection efficacy of both vaccines was evaluated by clinical signs, mortality rates, and virus shedding from oropharynx and cloaca of vaccinated chickens after challenge with HPAI A/CK/Korea/ES/03 (H5N1) virus. One dose of 128 hemagglutinin (HA) homologous H5N1 vaccine induced 100% protection in mortality and prevented viral shedding completely after lethal dose virus challenge, whereas one dose of 64 HA unit of heterologous H5N3 vaccine only induced 50% protection in mortality, and it did not prevent viral shedding. However, two doses at a 3-wk interval of 64 HA unit of heterologous H5N3 vaccine as well as one dose of 1024 HA unit of heterologous H5N3 vaccine induced 100% survival rate and could prevent viral shedding completely. Furthermore, we could differentiate the sera of infected birds from those of vaccinated birds by indirect immunofluorescent antibody test. These results suggest that heterologous neuraminidase H5N3 vaccine could be a useful tool for the control of H5N1 HPAI epidemic in poultry.