Aim This study was to examine the effect of ethyl acetate extract of shiitake mushroom on a human breast cancer line-MCF-7 cell in vitro. Method Cell cytotoxicity was examined using MTT method, Cell apoptosis was examined by AnnexinV-FITC/PI staining,Cell cycle analysis was determined by flow cytometry. Western blot analysis was used to determine Cyclin D_1、Cdk4、Bax and p21WAE1/CIP1 protein expression. Results Concentration-dependent cytotoxic effects of the extract were observed in MCF-7 human breast cancer cell lines. Pro-apoptotic proteins (Bax, p21WAE1/CIP1)were up-regulated while proteins involved in cell cycle(Cyclin D_1 and Cdk4)were down-regulated. Conclusion This study suggests that the anti-breast cancer activities might result from increased apoptosis and growth arrest