Eupolyphaga sinensis Walker is not only used as a food to enhance immunity, but is used as a traditional Chinese medicine and is known as the "preferred drug to regulate blood flow". Previous studies have reported its potential biological activities including anticoagulation, antithrombotic, liver protective effect and antitumor effects. Our results indicated that E. sinensis Walker 70% ethanol extract exhibited anti-tumor effects on S180 (murine sarcoma cell line) cells implanted mice. It effectively inhibited K562 (human chronic myeloid leukemia cell line) cells proliferation and induced G(2)-M phase arrest accompanying through up-regulation of cyclin B1, cdc2 and down-regulation of cyclin D1, cyclin E1, cdc25c and p53. In addition, it inhibited EGF secretion and EGFR kinase activity. Western blotting analysis indicated that it also inhibited the phosphorylation EGFR and activation of its downstream signaling molecules AKT and ERK. These results suggested that the antitumor mechanism of E. sinensis Walker involved altering the cell cycle and inhibiting EGFR phosphorylation in the EGFR signaling pathway.