Streptococcus pneumoniae resides on the mucosal surface of the upper respiratory tract and is ready to spread and trigger clinical diseases. Hence the vaccine that can eliminate the nasopharyngeal colonization was thought to be an ideal protective strategy against pneumococcal invasive diseases. Caseinolytic protease X (ClpX), a pneumococcal caseinolytic protease ATPase subunit, was shown to be a non-transmembrane protein by bioinformatics analysis. Consistent with the in silico prediction, the secretory expression of ClpX, instead of surface exposure, was further confirmed by flow cytometry and Western blot. Furthermore, ClpX was highly conserved in nine different serotypes of S. pneumoniae at both gene and protein concentrations. In addition, the anti-ClpX IgG antibody levels in human serum samples were much higher in healthy children, compared with pediatric patients, and displayed an age-related increase. Finally, ClpX protein antigen was introduced to BALB/c mice through a mucosal route, and protection against nasopharyngeal colonization and lethal infection caused by different S. pneumoniae serotypes was successfully elicited. Our findings suggest that ClpX is a potential candidate antigen that could be incorporated in pneumococcal protein vaccines.