Microbial transformations, especially the reduction and methylation of Se oxyanion, have gained significance in recent years as effective detoxification methods. Ganoderma lucidum is a typical Se enrichment resource that can reduce selenite to elemental Se and volatile Se metabolites under high selenite conditions. However, the detailed biological processes and reduction mechanisms are unclear. In this study, G. lucidum reduced selenite to elemental Se and further aggregated it into Se nanoparticles with a diameter of < 200 nm, simultaneously accompanied by the production of pungent, odorous, and volatile methyl-selenium metabolites. Tandem mass tag-based quantitative proteomic analysis revealed thioredoxin 1, thioredoxin reductase (NADPH), glutathione reductase, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, and cystathionine gamma-lyase as proteins involved in selenite reduction and methylation. Furthermore, the high expression of proteins associated with cell structures that prompted cell lysis may have facilitated Se release. The upregulation of proteins involved in the defense reactions was also detected, reflecting their roles in the self-defense mechanism. This study provides novel insights into the vital role of G. lucidum in mediating Se transformation in the biogeochemical Se cycle and contributes to the application of fungi in Se bioremediation. Display omitted ●Ganoderma lucidum played a vital role in mediating Se transformation and Se cycle in the environment.●Biogenic Se-nanoparticles occurred intracellularly and largely mediated by trxA, trxB and GSR.●The highly expressions of metE, CTH and trxB contributed to the production of 10 types of methyl-selenium metabolites.●The release of Se-nanoparticles in G. lucidum was by the looseness of cell wall and cell membrane structures.