Influenza A virus (IAV) is a deadly zoonotic pathogen that remains a burden to global health systems despite continuous vaccinations, indicating the need for an improved vaccine strategy. In this work, we constructed a new recombinant influenza vaccine using Bacillus subtilis spores expressing M2e-FP protein (RSM2eFP) and assessed its potency and efficacy in BALB/c mouse immunized via aerosolized intratracheal inoculation (i.t.) or intragastric (i.g.) administration. Immunization via i.t. route conferred 100 % protection against 20 × LD50 A/PR/8/34 (H1N1) virus compared with only 50 % via the i.g. route. Even when challenged with 40 × LD50 virus, the RSM2eFP vaccine immunized via i.t. provided 80 % protection. Consistently, i.t. inoculation of RSM2eFP spore vaccine induced a stronger lung mucosal immune response and a greater cellular immune response than i.g. administration, as indicated by the high production of IgG and SIgA. In addition, the RSM2eFP spore vaccine diminished the yield of infectious virus in the lung of mice immunized via i.t. These results suggest that i.t. immunization of the RSM2eFP spore vaccine may be a promising strategy for the development of mucosal vaccines against IAV infections.