The microbial reduction of selenite to elemental selenium nanoparticles (SeNPs) is thought to be an effective detoxification process of selenite for many bacteria. In this study, Metasolibacillus sp. ES129 and Oceanobacillus sp. ES111 with high selenite reduction efficiency or tolerance were selected for systematic and comparative studies on their performance in selenite removal and valuable SeNPs recovery. The kinetic monitoring of selenite reduction showed that the highest transformation efficiency of selenite to SeNPs was achieved at a concentration of 4.24 mM for ES129 and 4.88 mM for ES111. Ultramicroscopic analysis suggested that the SeNPs produced by ES111 and ES129 had been formed in cytoplasm and subsequently released to extracellular space through cell lysis process. Furthermore, the transcriptome analysis indicated that the expression of genes involved in bacillithiol biosynthesis, selenocompound metabolism and proline metabolism were significantly up-regulated during selenite reduction, suggesting that the transformation of selenite to Se0 may involve multiple pathways. Besides, the up-regulation of genes associated with nucleotide excision repair and antioxidation-related enzymes may enhance the tolerance of bacteria to selenite. Generally, the exploration of selenite reduction and tolerance mechanisms of the highly selenite-tolerant bacteria is of great significance for the effective utilization of microorganisms for environmental remediation. The concentration margin between dietary deficiency (< 40 μg/day) and toxic level (> 400 μg/day) in humans is very narrow, and excessive intake of Se may cause serious health problems. The reduction of toxic and soluble selenite to nontoxic and insoluble elemental selenium is an effective strategy to remediate selenium pollution and recover valuable selenium resources. In this study, strains ES111 and ES129 with high selenite tolerance or reduction efficiency were studied on their performance in selenite removal and valuable SeNPs recovery. This study revealed the selenite reduction and tolerance mechanism of the highly selenite-tolerant bacteria, which provided potential applications in bioremediation of selenite pollution.