Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.