Selenite biotransformation by microorganisms is an effective detoxification and assimilation process. However, current knowledge of the molecular mechanisms of selenite reduction remains circumscribed. Here, the reduction of Se(IV) by a highly selenite-resistant Bacillus sp. SL (up to 50mM) was systematically analyzed, and the molecular mechanisms of selenite reduction were investigated. Remarkably, 10mM selenite was entirely transformed by the strain SL within 20h, demonstrating a faster conversion rate compared to other microorganisms. Furthermore, glutathione (GSH) and exopolysaccharides (EPS) changes were also monitored during the process. Transcriptomic analysis revealed that the genes of ferredoxin-sulfite oxidoreductase (6.82) and sulfate adenylyltransferase (6.32) were significantly upregulated, indicating that the sulfur assimilation pathway is the primary reducing pathway involved in selenite reduction by strain SL. Moreover, key genes associated with NAD(P)/FAD-dependent oxidoreductases and thioredoxin were significantly upregulated. The reduction of Se(IV) was mediated by multiple pathways in strain SL. To our knowledge, this is the initial report to identify the involvement of sulfur assimilation pathway in selenite reduction for bacillus, which is rare in aerobic bacteria.