Streptococcus mutans is considered to be the most cariogenic organism. Carolacton, isolated from the myxobacterium Sorangium cellulosum, shows the ability to disturb S. mutans biofilm viability that makes it a potential anti-biofilm drug. However, the molecular mechanism of carolacton remains to be elucidated. In order to use proteomics to characterize the effect of carolacton, we constructed a 2DE-based proteome reference map of the cytoplasmic and extracellular proteins for S. mutans in the present study. In total, 239 protein spots representing 192 different cytoplasmic proteins were identified by MALDI-TOF MS and PMF. This represents the highest number of identified proteins so far for S. mutans UA159 in the pI range of 4-7 and would benefit further research on the physiology and pathogenicity of this strain. Based on the constructed reference map, the inhibitory effects of carolacton on S. mutans biofilm and planktonic-growing cells were investigated. The results of the comparative proteome analysis indicate that carolacton exerts its inhibitory effects by disturbing the peptidoglycan biosynthesis and degradation and thereby causes damages to the integrity of the cell envelope, leading ultimately to cell death.