Proteins and ribonucleoproteins containing a nuclear export signal (NES) assemble with the exportin Xpo1p (yeast CRM1) and Gsp1p-GTP (yeast Ran-GTP) in the nucleus and exit through the nuclear pore complex. In the cytoplasm, Yrb1p (yeast RanBP1) displaces NES from Xpo1p. Efficient export of NES-cargoes requires Yrb2p (yeast RanBP3), a primarily nuclear protein containing nucleoporin-like phenylalanine-glycine (FG) repeats and a low-affinity Gsp1p-binding domain (RanBD). Here, we show that Yrb2p strikingly accelerates the association of Gsp1p-GTP and NES to Xpo1p. We have solved the crystal structure of the Xpo1p-Yrb2p-Gsp1p-GTP complex, a key assembly intermediate that can bind cargo rapidly. Although the NES-binding cleft of Xpo1p is closed in this intermediate, our data suggest that preloading of Gsp1p-GTP onto Xpo1p by Yrb2p, conformational flexibility of Xpo1p, and the low affinity of RanBD enable active displacement of Yrb2p RanBD by NES to occur effectively. The structure also reveals the major binding sites for FG repeats on Xpo1p. Display omitted •Yrb2p increases the association rate of Gsp1p-GTP and NES with Xpo1p•The crystal structure of Xpo1p-Yrb2p-Gsp1p-GTP complex is determined•Yrb2p primes Xpo1p and Gsp1p for rapid loading of NES by an allosteric mechanism•The structure also reveals the major FG-repeat binding sites on Xpo1p Koyama et al. now look at how the Ran-binding protein Yrb2p (yeast RanBP3) promotes nuclear export of proteins and ribonucleoproteins. Yrb2p strikingly increases the rate of cargo loading onto the nuclear export receptor Xpo1p (yeast CRM1) through an allosteric mechanism.