Respiratory tract coinfections, specifically involving influenza A virus (IAV) and Streptococcus pneumoniae (S. pneumoniae), remain a major health problem worldwide. Secondary bacterial pneumonia is a common complication and an important cause of mortality related to seasonal and pandemic influenza infections. Vaccination is a basic control strategy against influenza and S. pneumoniae. The fusion protein DnaJ-ΔA146Ply is a vaccine candidate which can induce immune responses against pneumococcal infections via mucosal and subcutaneous immunization in mice. In the present study, we established a co-infection model using mouse-adapted laboratory strains of IAV (PR8) and S. pneumoniae (19F) in mice intranasally and subcutaneously immunized with DnaJ-ΔA146Ply. Our results showed that vaccinated mice suffered decreased weight loss compared with control mice. The survival rates were higher in intranasally and subcutaneously immunized mice than in control mice. In addition, the bacterial loads in nasal washes and lung homogenates were lower in vaccinated mice than in control mice. Furthermore, lung damage was alleviated in vaccinated mice compared with control mice, with less broken alveoli and less proinflammatory cytokine production. Taken together, these results indicate that vaccination with DnaJ-ΔA146Ply shows protective potential against influenza and S. pneumoniae co-infection in mice.