Updated publication reference for PubMed record(s): 29435015. The breast cancer incidence has been increasing in China, with the earlier age of onset compared with Western countries. Traditional Chinese medicine has been provided as one of the major source of anti-cancer drugs. Ginseng is one of the most common traditional medicines in China. Ginsenosides, the saponins in the plant Panax (ginseng) are the major active components responsible for their chemopreventive effects from cancer. However, the mechanisms by which ginsenosides exert their anti-cancer effect remain elusive. By combining TMT-based quantitation with TiO2-based phosphopeptide enrichment, we performed a quantitative analysis of the changes of the phosphoproteomes in ginsenoside Rg3-treated breast cancer MDA-MB-231 cells. We were able to quantitate 5,140 phosphorylation sites on 2,041 phosphoproteins. Our data show that the phosphorylation status of 13 sites was changed in MDA-MB-231 cells upon Rg3 treatment. The perturbed phosphoproteins are CPSF7, EEF2, HIRIP3, MAGED2, MPRIP, MYCBP2, PAWR, PPP1R12A, RANBP2, SEPT9, TMPO, and UFL1. These proteins are involved in various biological processes, including protein synthesis, cell division, and inhibition of NF-κB signaling. Our study revealed that Rg3 exerts its anti-cancer effects through a combination of different signaling pathways.