Simian immunodeficiency virus (SIV) and human immunodeficiency virus 2 (HIV-2) display unique ability to infect nondividing target cells. Viral protein X (Vpx) of HIV-2/SIV is known to be involved in the nuclear import of viral genome in nondividing cells, but the mechanism remains poorly understood. In the present investigation for the first time we provide evidence that Vpx of SIVsmPBj1.9 physically interacts with human nucleoporin 153 (Nup153), which is known to provide a docking site for protein-cargo complexes at the nuclear pore complex (NPC). Results from superresolution-structured illumination microscopy studies reveal that Vpx interaction with NPC-associated Nup153 is critical for its efficient nuclear translocation. Virion-associated MAPK/ERK-2-mediated phosphorylation of Vpx plays a critical role in its interaction with human Nup153 and this interaction was found to be evolutionarily conserved in various SIV isolates and HIV-2. Interestingly, MAPK/ERK-2 packaging defective SIV failed to promote the efficient nuclear import of viral genome and suggests that MAPK/ERK-2-mediated Vpx phosphorylation is important for its interaction with Nup153, which is critical for lentiviruses to establish infection in nondividing target cells. Together, our data elucidate the mechanism by which Vpx orchestrates the challenging task of nuclear translocation of HIV-2/SIV genome in nondividing target cells.