Korean traditional medicine, HangAmDan (HAD) was developed in 1996 for anti-tumor purpose, and has shown positive results in clinical case studies. Recently, HAD has been recently modified as HAD-B to increase therapeutic effects. Our present study focused on the effect of HAD-B on proteome alteration in human colon cancer cells with highly aggressive invasiveness. Before the proteome assessment, invasiveness of 4 human colon cancer cell lines, SNU-407, SNU-C4, LoVo, and DLD-1 have been determined. Among cell lines tested, DLD-1 showed higher invasiveness, and was selected for investigating proteome affected by HAD-B. HAD-B reduced invasiveness of DLD-1, but it did not lead to either synergistic effect with 5-fluorouracil or apoptosis. Phosphorylation of eukaryotic initiation factor 4E (eIF-4E) binding protein 1 (4EBP1) and mammalian target of rapamycin (mTOR) in DLD-1 was increased by HAD-B treatment whereas level of AKT phosphorylation was reduced. Furthermore, expression of eIF-5A-2 and the balance between tissue inhibitor of metalloproteinases-1 (TIMP-1) and matrix metalloproteinase 9 (MMP9) were deranged in DLD-1 after treatment of HAD-B. In conclusion, overall results from our proteome assessment may provide useful information on how HAD-B suppresses proliferation and invasion of human colon cancer cells.