Streptococcus pneumoniae is a major human respiratory pathogen causing considerable morbidity and mortality worldwide. In order to better understand the pathogenesis of S. pneumoniae, we employed SDS-PAGE combined with LC-MS/MS analysis and in-solution digestion coupled with 2D-LC-MS/MS to obtain the whole-cell proteome of the bacterium. Among the identified 1,210 proteins, 345 proteins were annotated for cellular components, 613 for biological processes, and 421 for molecular functions. Important virulence-associated surface proteins such as Eno, ZmpB, and PrtA were identified. Classification analysis and protein-protein interaction map revealed that these identified proteins are involved in many biological processes including protein biosynthesis, protein folding and proteolysis, cell cycle, or regulation and carbohydrate metabolism. These data represent a comprehensive reference map of S. pneumoniae proteome, providing a useful source for further analysis of the virulence factors and the regulatory network involved in the pathogenesis of the bacterium.