Anti-adenovirus serotype 5 antibodies are capable of neutralizing adenovirus serotype 5-based vaccines. In mice and guinea pigs, intranasal delivery of adenovirus serotype 5-based vaccine bypasses induced adenovirus serotype 5 preexisting immunity, resulting in protection against species-adapted Ebola virus challenge. In this study, nonhuman primates were vaccinated with adenovirus serotype 5-based vaccine either intramuscularly or via the airway route (intranasally/intratracheally) in the presence or absence of adenovirus serotype 5 preexisting immunity. Immune responses were evaluated to determine the effect of both the vaccine delivery route and preexisting immunity before and after a lethal Ebola virus (Zaïre strain Kikwit 95) challenge. Intramuscular vaccination fully protected nonhuman primates in the absence of preexisting immunity, whereas the presence of preexisting immunity abrogated vaccine efficacy and resulted in complete mortality. In contrast, the presence of preexisting immunity to adenovirus serotype 5 did not alter the survival rate of nonhuman primates receiving the adenovirus serotype 5-based Ebola virus vaccine in the airway. This study shows that airway vaccination with adenovirus serotype 5-based Ebola virus vaccine can efficiently bypass preexisting immunity to adenovirus serotype 5 and induce protective immune responses, albeit at lower efficacy than that using an intramuscular vaccine delivery route.