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The antiproliferative and apoptosis-inducing activities of an acidic polysaccharides fraction from Pholiota dinghuensis Bi mycelium (PDP-3) against human breast cancer MCF-7 cells were investigated. As a result, PDP-3 displayed a significant prevention of cell growth towards MCF-7 cells, with an EC50 value of 38.35 µg/mL. Furthermore, PDP-3 induced significant apoptosis towards MCF-7 cells. From Western blot results, up-regulated p21 and down-regulated cyclin D1, CDK4 and PCNA, up-regulated Bax and down-regulated Bcl-2, caspase-9 and caspase-3 were found in the protein expressions of MCF-7 cells. In MCF-7 cells treated with PDP-3, down-regulated TRAF2 and up-regulated ASK1, phosphorylated of p38 and p53 were also found. Notably, PDP-3 treatment represented a significant decrease in both nuclear and cytoplasmic expression of ERα in MCF-7 cells. Therefore, p38/MAPK signal transduction pathway was the potential mechanism of PDP-3 in regulating both cell proliferation and apoptosis in oestrogen-dependent human breast cancer MCF-7 cells.