Updated project metadata. Cargo molecules which exceed the passive diffusion limit of ~5nm require active translocation to cross the nuclear pore complex (NPC). Such a facilitated transport is achieved by specialized nuclear transport receptors (NTRs), which are classified according to their shuttling direction. Exportins bind their cargo in a RanGTP-dependent manner inside the nucleus and release it in the cytoplasm. Importins, on the other hand, bind their cargo without RanGTP and transfer it in the opposite direction. CRM1/Xpo1 is one of the best-characterized NTRs mostly due to the availability of specific inhibitors, like leptomycin B, which allowed cargo validation in vivo. Such inhibitors have not been characterized for other NTRs, thus analysis of those lagged behind. Here, we developed nanobodies to specifically target exportin 7/Xpo7 and block its transport pathway. Moreover, in-depth MS/MS analysis of Xpo7 under nuclear (+RanGTP) and cytoplasmic conditions (-RanGTP) revealed novel binders including ~200 potential export substrates, but also ~30 nuclear import cargoes. Enrichment factors of a putative cargo were calculated using the iBAQ strategy to quantify detectable proteins in the input as well as in the import or export mimicking material. Validation of selected cargo molecules was then accomplished by anti-Xpo7 nanobodies causing cargo mislocalization when transfected into cultured cells. Collectively, the data establish Xpo7 as a bidirectional NTR with a broad substrate specificity.