Pneumococcal asymptomatic colonization of the respiratory tracts is a major risk for invasive pneumococcal disease. We have previously shown that pneumococcal wall teichoic acid (WTA) was involved in pneumococcal infection of sepsis and adherence to epithelial and endothelial cells. In this study, we investigated the contribution of pneumococcal WTA to bacterial colonization and dissemination in murine models. The result showed that nasopharynx colonizing D39 bacterial cells have a distinct phenotype showing an increased exposure of teichoic acids relative to medium-grown bacteria. The WTA-deficient mutants were impaired in their colonization to the nasopharynx and lungs, and led to a mild inflammation in the lungs at 36 h post-inoculation. Pretreatment of the murine nares with WTA reduced the ability of wild type D39 bacteria to colonize the nasopharynx. In addition, the WTA-deficient strain was impaired in its ability to invade the blood and brain following intranasal administration. WTA-deficient D39 strain was reduced in C3 deposition but was more susceptible to the killing by the neutrophils as compared with its parent strain. Our results also demonstrated that the WTA enhanced pneumococcal colonization and dissemination independently of the host strains. These results indicate that WTA plays an important role in pneumococcal pathogenesis, both in colonization and dissemination processes.