Abstract: Objective To investigate the role and signaling mechanisms of calcium-activated neutral protease (CANP) involved in cell migration and proteolysis of FAK protein induced by estradiol (E2) in breast cancer. Methods Wound healing assay was carried out to evaluate cell migration in human breast cancer cell line MCF-7. The effects of E2 or epidermal growth factor (EGF) on the truncation of focal adhesion kinase (FAK) protein were detected by Western blotting.A specific inhibitor of CANP, Calpeptin, and an intracellular calcium chelator, BAPTA/AM,were applied to pre-treat cultured cells to evaluate their influences on the E2-stimulated cell migration and FAK proteolysis. Results E2 was able to markedly induced MCF-7 cell migration and FAK proteolysis. This effect can be significantly inhibited by Calpeptin BAPTA pre-treatment. E2 also induced CANP1 autolysis, which might be significantly weakened or blocked by Calpeptin or BAPTA. Conclusion E2 induced breast cancer cell migration and FAK proteolysis(an important factor in E2-induced cell migration) through intracellular Ca2+/ the CANP signal pathway.