<p>Central nervous system (CNS) macrophages play key roles in viral neuropathogenesis and immune surveillance, yet their trafficking and trafficking dynamics between the CNS and peripheral lymphoid tissues remain poorly understood. To address this gap, we used intracisternal (i.c.) injection of fluorescent-superparamagnetic iron oxide nanoparticles (SPION) in SIV-infected macaques that we found labeled CNS CD68⁺CD163⁺CD206⁺ perivascular, meningeal, and choroid plexus (CP) macrophages. SPION⁺CD163⁺ macrophages were also detected in the optic nerves and cribriform area—potential routes of macrophage egress from the CNS. CD163⁺SPION⁺ macrophages labeled in the CNS were identified in the deep cervical lymph node (dCLN) and dorsal root ganglia (DRG). SPION⁺ CD163⁺ macrophages appeared in both the CNS and the periphery as early as 24&#xa0;h post i.c. inoculation, decreasing over time in the CNS of non-infected animals but accumulating with SIV infection. A trend toward greater numbers of SPION ⁺ macrophages that traffic out of the CNS was observed in non-infected animals, a pattern not seen with SIV infection. Productively infected SPION⁺ cells were found in the CNS and dCLN of infected animals 7–28&#xa0;days post i.c. injection. These findings support a model in which SPION ⁺ macrophages, some of which harbor virus, traffic between the CNS and peripheral lymphoid tissues. This is discussed with regard to HIV infection of the CNS and reseeding of the periphery with CNS virus.</p>