Phenyl-thiazolylurea-sulfonamides compound 1 (ptsc1) is a new and efficient bacterial inhibitor. Although the inhibitor has antibacterial activity against several model bacteria, the details of its mechanism in Streptococcus pneumoniae (S. pneumoniae) have not been revealed. In the current study, we found that ptsc1 can significantly inhibit the growth of S. pneumoniae. To further investigate the molecular mechanism of its antibacterial effects, quantitative proteomics was performed to identify differential expressed proteins in S. pneumoniae. Compared with the normal group, the expression levels of 38 proteins were upregulated, whereas those of 88 proteins were downregulated (> 1.3 fold-change) in the experimental group (P < 0.05). KEGG pathways analysis revealed that ptsc1 regulated proteins were mainly involved in carbohydrate metabolism, DNA replication and repair, pyrimidine metabolism, fatty acid biosynthesis and oxidative phosphorylation pathways. The results of GO enrichment analysis showed that the inhibitor could lead to dysregulation of bacterial translation and energy metabolism, and cause intracellular oxidative stress.