S-glutathionylation is an important post-translational modification (PTM) process that targets the protein cysteine thiol by the addition of glutathione (GSH). This modification can prevent proteolysis from over-oxidation of protein cysteine residue during the condition of oxidative or nitrosative stress. Recent studies suggest that protein S-glutathionylation plays an essential role in control of cell-signaling pathways by affecting the protein function in bacteria and even humans. In this study, we investigated the impacts of S-glutathionylation on physiological regulation within Streptococcus mutans, the primary etiological agent of human dental caries. To determine S-glutathionylated proteins in bacteria, the Cys-reactive isobaric reagents iodoTMT (iodoacetyl Tandem Mass Tag) were used to label the S-glutathionylated Cys sites and anti-TMT antibody conjugated resins were used to enrich the modified peptides. Proteome profiling identified a total of 357 glutathionylated cysteines sites on 239 proteins. Functional enrichment analysis indicated that these S-glutathionylated proteins were involved in diverse important biological processes, such as pyruvate metabolism and glycolysis