[en] Highlights: • Shotgun proteomics revealed Se detoxification systems in Stenotrophomonas bentonitica. • Proteins for transport, reduction and volatilization of Se(IV) were identified. • A key role of thiol-containing enzymes in Se(IV) reduction is suggested. • Biotransformation from Se(0)NPs to m-Se and t-Se nanostructures was confirmed. • A cellular model for Se(IV) response in Stenotrophomonas bentonitica was proposed. The widespread use of selenium (Se) in technological applications (e.g., solar cells and electronic devices) has led to an accumulation of this metalloid in the environment to toxic levels. The newly described bacterial strain Stenotrophomonas bentonitica BII-R7 has been demonstrated to reduce mobile Se(IV) to Se(0)-nanoparticles (Se(0)NPs) and volatile species. Amorphous Se-nanospheres are reported to aggregate to form crystalline nanostructures and trigonal selenium. We investigated the molecular mechanisms underlying the biotransformation of Se(IV) to less toxic forms using differential shotgun proteomics analysis of S. bentonitica BII-R7 grown with or without sodium selenite for three different time-points. Results showed an increase in the abundance of several proteins involved in Se(IV) reduction and stabilization of Se(0)NPs, such as glutathione reductase, in bacteria grown with Se(IV), in addition to many proteins with transport functions, including RND (resistance-nodulation-division) systems, possibly facilitating Se uptake. Notably proteins involved in oxidative stress defense (e.g., catalase/peroxidase HPI) were also induced by Se exposure. Electron microscopy analyses confirmed the biotransformation of amorphous nanospheres to trigonal Se. Overall, our results highlight the potential of S. bentonitica in reducing the bioavailability of Se, which provides a basis both for the development of bioremediation strategies and the eco-friendly synthesis of biotechnological nanomaterials.