Many pathogens infect animal hosts via the nasal route. Thus, understanding how vaccination stimulates early nasal immune responses is critical for animal and human health. Vaccination is the most effective method to prevent disease outbreaks in farmed fish. Nasal vaccination induces strong innate and adaptive immune responses in rainbow trout and was shown to be highly effective against infectious hematopoietic necrosis (IHN). However, direct comparisons between intranasal, injection and immersion vaccination routes have not been conducted in any fish species. Moreover, whether injection or immersion routes induce nasal innate immune responses is unknown. The goal of this study is to compare the effects of three different vaccine delivery routes, including intranasal (IN), intramuscular (i.m.) injection and immersion (imm) routes on the trout nasal innate immune response. Expression analyses of 13 immune-related genes in trout nasopharynx-associated lymphoid tissue (NALT), detected significant changes in immune expression in all genes analyzed in response to the three vaccination routes. However, nasal vaccination induced the strongest and fastest changes in innate immune gene expression compared to the other two routes. Challenge experiments 7 days post-vaccination (dpv) show the highest survival rates in the IN- and imm-vaccinated groups. However, survival rates in the imm group were significantly lower than the IN- and i.m.-vaccinated groups 28 dpv. Our results confirm that nasal vaccination of rainbow trout with live attenuated IHNV is highly effective and that the protection conferred by immersion vaccination is transient. These results also demonstrate for the first time that immersion vaccines stimulate NALT immune responses in salmonids.