Fe65 is characterized as an adaptor precursor (APP) through its PID2 element, as well as with the other members of the APP protein family. With the serum- and glucocorticoid-induced kinase 1 (SGK1) substrate specificity information, we found that the putative site of phosphorylation in Fe65 by SGK1 is present on its $Ser^{566}$ residue in $^{560}CRVRFLSFLA^{569}$(X60469). Thus, we demonstrated that Fe65 and the fluorescein-labeled Fe65 peptide $FITC-^{560}CRVRFLSFLA^{569}$ are phosphorylated in vitro by SGK1. Phosphorylation of the $Ser^{566}$ residue was also demonstrated using a $Ser^{566}$ phospho-specific antibody. The phospho Fe65 was found mainly in the nucleus, while Fe65 S556A mutant was localized primarily to the cytoplasm. Therefore, these data suggest that SGK1 phosphorylates the $Ser^{566}$ residue of Fe65 and that this phosphorylation promotes the migration of Fe65 to the nucleus of the cell.