Selenoprotien W (SelW) is an important selenoprotein that has the priority to be stored in brain when selenium is deficiency. However, the biological function and mechanisms of SelW in brain remain unclear. In this study, human SelW gene was cloned, site-directedly mutated and inserted into the "bait" plasmid. An interactive protein of SelW, prosaposin (PSAP), was discovered by screening the human fetal brain cDNA library using the yeast two-hybrid system. To verify the protein-protein interaction, two FRET methods, sensitized emission and acceptor bleaching, were performed respectively in HEK293T cells. Both assays confirmed the interaction between SelW′ and PSAP. Then the expression vector of SelW′ was constructed and SelW was overexpressed in E. coli. Pull-down assay was carried out by using the purified SelW′, and exogenously direct interaction between SelW′ and PSAP was also verified. Finally, co-IP method was applied to successfully verify the endogenous interaction between the two proteins in the brain tissues of Kunming mice. All these results show that SelW interacts with PSAP directly both in vivo and in vitro. SelW may play a key role in the brain development and neurodegenerative disease formation by interacting with PASP.