During infection, the common respiratory tract pathogen Streptococcus pneumoniae encounters several environmental conditions, such as upper respiratory tract, lung tissue, and blood stream, etc. In this study, we examined the effects of blood on S. pneumoniae protein expression using a combination of highly sensitive 2-dimensional electrophoresis (DE) and MALDI-TOF MS and/or LC/ESI-MS/MS. A comparison of expression profiles between the growth in THY medium and THY supplemented with blood allowed us to identify 7 spots, which increased or decreased two times or more compared with the control group: tyrosyl-tRNA synthetase, lactate oxidase, glutamyl-aminopeptidase, L-lactate dehydrogenase, cysteine synthase, ribose-phosphate pyrophosphokinase, and orotate phosphoribosyltransferase. This global approach can provide a better understanding of S. pneumoniae adaptation to its human host and a clue for its pathogenicity.