ABSTRACTMicroorganisms capable of converting toxic selenite into elemental selenium (Se0) are considered an important and effective approach for bioremediation of Se contamination. In this study, we investigated the mechanism of reducing selenite to Se0and forming Se nanoparticles (SeNPs) by food-grade probioticLactobacillus caseiATCC 393 (L. caseiATCC 393) through proteomics analysis. The results showed that selenite added during the exponential growth period of bacteria has the highest reduction efficiency, and 4.0 mM selenite decreased by nearly 95% within 72 h and formed protein-capped-SeNPs. Proteomics analysis revealed that selenite induced a significant increase in the expression of glutaredoxin, oxidoreductase, and ATP binding cassette (ABC) transporter, which can transport glutathione (GSH) and selenite. Selenite treatment significantly increased theCydCandCydD(putative cysteine and glutathione importer, ABC transporter) mRNA expression level, GSH content, and GSH reductase activity. Furthermore, supplementation with an additional GSH significantly increased the reduction rate of selenite, while GSH depletion significantly inhibited the reduction of selenite, indicating that GSH-mediated Painter-type reaction may be the main pathway of selenite reduction inL. caseiATCC 393. Moreover, nitrate reductase also participates in the reduction process of selenite, but it is not the primary factor. Overall,L. caseiATCC 393 effectively reduced selenite to SeNPs by GSH and nitrate reductase-mediated reduction pathway, and the GSH pathway played the decisive role, which provides an environmentally friendly biocatalyst for the bioremediation of Se contamination.IMPORTANCEDue to the high solubility and bioavailability of selenite, and its widespread use in industrial and agricultural production, it is easy to cause selenite to accumulate in the environment and reach toxic levels. Although the bacteria screened from special environments have high selenite tolerance, their safety has not been fully verified. It is necessary to screen out strains with selenite-reducing ability from nonpathogenic, functionally known, and widely used strains. Herein, we found food-grade probioticL. caseiATCC 393 effectively reduced selenite to SeNPs by GSH and nitrate reductase-mediated reduction pathway, which provides an environmentally friendly biocatalyst for the bioremediation of Se contamination.