DNA immunization is a potential vaccination strategy for neonates and infants. We tested the ability of a prototype DNA vaccine against influenza virus to prime lasting immunity when administered to newborn non-human primates. Neonatal DNA vaccination triggered virus-specific and neutralizing antibodies of titers and persistence depending on the vaccine dose. Subsequent exposure to influenza virus, revealed significantly increased recall responses in the baboons vaccinated with DNA during the neonatal stage. The humoral and cellular responses were enhanced in the baboons primed with DNA vaccine as neonates. Thus, neonatal DNA vaccination of non-human primates triggered immune memory that persisted beyond infancy.