Protection of chickens against avian influenza (AI) is mostly attributed to production of antibodies against the viral glycoprotein hemagglutinin, whereas less is known about the protective role of antibodies to the other surface glycoprotein neuraminidase (NA). Therefore, vaccines encoding NA antigen (e.g., DNA and alphavirus-based virus like replicon particles (VRP)) or baculovirus-expressed recombinant NA (rN2) were tested for their ability to protect against highly pathogenic AI (HPAI) in chickens. Vaccination with A/Pheasant/Maryland/4457/93 (Ph/MD) rN2 protein produced significantly higher levels of NA-inhibition (NI) activity and 88% protection from HPAI H5N2 challenge than vaccination with Ph/MD N2 DNA (25% protection). Vaccination with Ph/MD N2 VRP a minimum of two times also produced high levels of NI activity and protection against HPAI challenge (63% protection). Vaccination with VRP encoding an N2 gene that was genetically distant from the challenge virus N2 failed to protect chickens. Vaccines producing higher levels of NI activity conferred partial protection, but failed to affect viral shedding. Consideration of the homology between vaccine and challenge virus isolate NA genes may provide improved immunity if high levels of NI activity are obtained.