ABSTRACT Streptococcus pneumoniae two-component regulatory systems (TCS) enable adaptation and ensure its maintenance in host environments. This study deciphers the impact of the TCS08 on pneumococcal gene expression and its role in metabolic and pathophysiological processes. Transcriptome analysis and real-time PCR demonstrated a regulatory effect of the TCS08 on genes involved mainly in environmental information processing, intermediary metabolism, and colonization by S. pneumoniae D39 and TIGR4. Striking examples are genes of the fatty acid biosynthesis, arginine-deiminase system, and psa operon encoding the manganese ABC transport system. In silico analysis confirmed that TCS08 is homologous to Staphylococcus aureus SaeRS and a SaeR-like binding motif is displayed in the promotor region of pavB , the upstream gene of the tcs08 operon encoding a surface-exposed adhesin. Indeed, PavB is regulated by the TCS08 as confirmed by immunoblotting and surface abundance assays. Similarly, Pilus-1 of TIGR4 is regulated by TCS08. Finally, in vivo infections using the acute pneumonia and sepsis models showed a strain dependent effect. Loss of function of HK08 or TCS08 attenuated D39 virulence in lung infections. The RR08 deficiency attenuated TIGR4 in pneumonia, while there was no effect on sepsis. In contrast, lack of HK08 procured a highly virulent TIGR4 phenotype in both pneumonia and sepsis infections. Taken together, these data indicate the importance of TCS08 in pneumococcal fitness to adapt to the milieu of the respiratory tract during colonization. IMPORTANCE Streptococcus pneumoniae interplays with its environment by using 13 two-component regulatory systems and one orphan response regulator. These systems are involved in the sensing of environmental signals thereby modulating pneumococcal pathophysiology. This study aimed to understand the functional role of genes subject to control by the TCS08. The identified genes play a role in transport of compounds such as sugars or amino acids. In addition, the intermediary metabolism and colonization factors are modulated by TCS08. Thus, TCS08 regulates genes involved in maintaining pneumococcal physiology, transport capacity and adhesive factors to enable optimal colonization, which represents a prerequisite for invasive pneumococcal disease.