Breast cancer is the most commonly diagnosed cancer among women in Western countries. Currently, there is no effective therapy for malignant estrogen-independent breast cancer. We have screened 38 species of edible mushroom on human estrogen-receptor positive (MCF-7) and estrogenreceptor negative (MDA-MB-231, BT-20) breast cancer cells to select potential agents with broad-spectrum antitumor activity against breast cancer cells. Water-based extracts of three mushroom species, Coprinellus sp., Coprinus comatus, Flammulina velutipes (CME, CCE and FVE respectively), were identified as novel anti-breast cancer agents. The antitumor activities include: 1) marked growth inhibition of both ER + and ERbreast cancer cells; 2) induction of rapid apoptosis on both ER + and ERcells; 3) significant inhibition of MCF-7 tumor colony formation in vitro. The antiproliferative and cytotoxic activities of the three mushroom extracts were dose-dependent, regardless of the hormone receptor status of the cancer cells. The degree of produced cytotoxicity on ERbreast cancer cells was very high, while the IC 50 of mushroom extract CME was found to be as low as 40 μg/ml on MDA-MB-231 cells and the IC 50 of mushroom extract FVE was only 30 μg/ml on BT-20 cells. More interestingly, mushroom extracts CME and FVE induced an exceptionally rapid apoptosis on MCF-7 and MDA-MB-231 detected by Annexin V-FITC within 2 h of treatment and DNA fragment endlabeling assay (TUNEL) in 5 h of treatment. Anchorageindependent growth assays indicated that the MCF-7 tumor colony formation rate was reduced by 60% in CCE-and CME-treated cells and nearly completely inhibited (99%) by FVE treatment. These results suggest that mushroom species Coprinus comatus, Coprinellus sp. and Flammulina velutipes contain potent antitumor compounds for breast cancer. Our finding is important due to the lack of chemotherapeutic and chemopreventive agents for ERhuman breast cancer.