Competence in the pathogenic bacterium Streptococcus pneumoniae (S. pneumoniae) is a developmental genetic program that is key for natural genetic transformation and consequently bacterial horizontal gene transfer. Phosphoproteomic studies have revealed that protein phosphorylation on serine, threonine and tyrosine residues is a widespread regulatory post-translational modification in bacteria. In this study, we performed quantitative proteomic and phosphoproteomic analyses on S. pneumoniae as a function of competence induction. To calculate peptide abundance ratios between non-competent and competent samples we used dimethyl-tag labeling. Titanium dioxide (TiO2) beads were used for phosphopeptide enrichment and samples were analysed by LC-MS/MS. Our proteome analysis covers approximatively 63 % of the total bacterial protein content, identifying 82 proteins with significantly different abundance ratios, including some not previously linked to competence. 248 phosphopeptides were identified including 47 having different abundance ratios. Notably, the proteins with a change in phosphorylation in competent cells are different from the proteins with a change in expression, highlighting different pathways induced by competence and regulated by phosphorylation. This is the first report that phosphorylation of some proteins is regulated during competence in Streptococcus pneumoniae, a key pathway for the bacteria to evade vaccines or acquire antibiotic resistance. SIGNIFICANCE: S. pneumoniae is a prominent model for the study of competence that governs the development of natural genetic transformation. The latter largely accounts for the spread of antibiotic resistance and vaccine evasion in pneumococcal isolates. Many proteins specifically expressed during competence have been identified and extensively studied. However, the potential contribution of post-translational modifications, and notably phosphorylation, during the development of competence has never been investigated. In this study, we used a quantitative phosphoproteomic approach to determine both the protein expression and the protein phosphorylation patterns. Comparison of these patterns allows to highlight a series of proteins that are differentially phosphorylated in the two conditions. This result opens new avenues to decipher new regulatory pathways induced by competence and that are potentially key for natural genetic transformation. Interfering with theses regulatory pathways could represent a promising strategy to combat antibiotic resistance in the future.