Nucleoprotein is highly conserved among each type of influenza viruses (A and B) and has received significant attention as a good target for universal influenza vaccine. In this study, we determined whether a recombinant adenovirus encoding nucleoprotein of type B influenza virus (rAd/B-NP) confers protection against influenza virus infection in mice. We also identified a cytotoxic T lymphocyte epitope in the nucleoprotein to determine B-NP-specific CD8 T-cell responses. We found that B-NP-specific CD8 T cells induced by rAd/B-NP immunization played a major role in protection following influenza B virus infection using CD8 knockout mice. To assess the effects of the administration routes on protective immunity, we immunized mice with rAd/B-NP via intranasal or intramuscular routes. Both groups showed strong NP-specific humoral and CD8 T-cell responses, but only intranasal immunization provided complete protection against both lineages of influenza B virus challenge. Intranasal but not intramuscular administration established resident memory CD8 T cells in the airway and lung parenchyma, which were required for efficient protection. Furthermore, rAd/B-NP in combination with rAd/A-NP protected mice against lethal infection with both influenza A and B viruses. These findings demonstrate that rAd/B-NP could be further developed as a universal vaccine against influenza.