Streptococcus pneumoniae is a respiratory pathogen, and mucosal immune response plays a significant role in the defense against pneumococcal infections. Thus, intranasal vaccination may be an alternative approach to current immunization strategies, and effective delivery systems to mucosal organism are necessary. In this study, BALB/c mice were immunized intranasally with chitosan-DNA nanoparticles expressing pneumococcal surface antigen A (PsaA). Compared to levels in mice immunized with naked DNA or chitosan-pVAX1, anti-PsaA IgG antibody in serum and anti-IgA antibody in mucosal lavages were elevated significantly in mice immunized with chitosan-psaA. The balanced IgG1/IgG2a antibody ratio in serum, enhanced gamma interferon (IFN-γ) and IL-17A levels in spleen lymphocytes, and mucosal washes of mice immunized with chitosan-psaA suggested that cellular immune responses were induced. Furthermore, significantly fewer pneumococci were recovered from the nasopharynx of mice immunized with chitosan-psaA than for the control group following intranasal challenge with ATCC 6303 (serotype 3). These results demonstrated that mucosal immunization with chitosan-psaA may successfully generate mucosal and systemic immune responses and prevent pneumococcal nasopharyngeal colonization. Hence, a chitosan-DNA nanoparticle vaccine expressing pneumococcal major immunodominant antigens after intranasal administration could be developed to prevent pneumococcal infections.