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To investigate the active compound and its mechanism of the anti-tumor effects of Auricularia polytricha, the polysaccharides were isolated and its effects on macrophages were tested. By alcohol-precipitation, DEAE-cellulose ion exchange, gel permeation chromatography and in vivo anti-tumor test on the sarcoma 180 tumor-bearing mice, a fraction of polysaccharides (namely APPIIA) with significant anti-tumor effect was isolated. APPIIA can enhance the neutral red phagocytosis of macrophages. It can also stimulate the nitric oxide (NO) release and cytokines secretion of macrophage in a dose-dependent manner. By quantitative RT-PCR method, it can be found that APPIIA can augment the mRNA transcription of inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6. The results of western blotting showed that it can also increased the protein expression of iNOS. These results suggest that APPIIA is one of effective anti-tumor agents and its anti-tumor effect may exhibit via the macrophage activation.