Ginseng is a representative herbal medicine in Asia and various pharmacological activities of ginsenoside Rd isolated from ginseng have been reported. However, anti-cancer activity and mechanism of ginsenoside Rd in HT29 colon cancer cell lines were not studied yet. We performed proteomic analysis through two-dimensional gel electrophoresis, MALDI-TOF/TOF-MS and database to identify altered protein induced by ginsenoside Rd treatment in HT29. We can identify fourteen proteins contributed to cell growth inhibition induced by Rd. Proteins involved in the inhibition of mitosis (Stathmin1, Microtubule-associated protein RP/EB family and Stratifin) were significantly up- and down-regulated. And proteins associated with apoptosis (Rho GDP dissociation inhibitor, Tropomyosin1 and Annexin5) were significantly changed. Furthermore, ginsenoside Rd in HT29 was involved in cytoprotection, DNA replication and repair, protein synthesis and degradation, metastasis and mutagenesis. It was supposed that ginsenoside Rd contributed to induce anti-cancer activity by complementary functions of these proteins in colon cancer cells.