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Mushrooms have become an important way to safely supply the body with the daily needs of organic selenium and they also possess remarkable medicinal properties. In this study, we examined the ability of the Pleurotus ostreatus mushroom to grow in selenium (Se) and its ability to accumulate and convert Se from inorganic form to organic form during growth. Additionally, we achieved the potential anticancer properties of mushroom extract in colon cancer cells using the CaCo-2 cell and the normal human colon mucosal epithelial cell line, NCM-460 cell line. Interestingly, Se-enriched mushroom extract (SME) showed a competitive regulation in colon cancer cell line; CaCo-2 cell line indicated by cell morphology, the number of survived cells, lactate dehydrogenase (LDH) production, and cell viability rate. Moreover, SME treatment regulates the expression profile of the cancer cell proliferation factor Raf-1 and pro-apoptotic related factors P53 and Caspase-3 Furthermore, the production of inflammatory-regulated cytokines, including interleukin 6 (IL-6) and IL-10, increased. At the same time, the level of produced tumor necrosis factor-alpha (TNF-α) markedly decreased in a dose and time-dependent of colon cancer-treated cells. Notably, the purified selenomethionine (SeMe) showed sufficient inhibition of colon cancer proliferation compared with the inorganic form of selenium (sodium selenite) via blocking the Raf/MEK/ERK signaling pathway. In addition, SeMe treatment also stimulated the production of IL-6 and IL-10 while decreasing the production of TNF-α, which plays a crucial role in the necrotic event. Meanwhile, the SeMe treatment showed a neglected cytotoxic effect in the normal colon epithelial cells. Collectively, these findings indicate that the fruiting bodies of Se-enriched mushrooms revealed anti-colon cancer activity via targeting Raf-1 signaling pathway and increasing the production of IL-6 and IL-10.