To examine the role of extracellular vesicles (EVs) in HIV neuropathogenesis, we isolated and characterized EVs from the brains of rhesus macaques, both with and without SIV induced CNS disease. Small RNA sequencing revealed significantly increased miR-21 levels in EVs from SIV encephalitic brains. In situ hybridization revealed increased miR-21 expression in neurons and macrophage/microglial cells/nodules during SIV induced CNS disease. In vitro culture of macrophages revealed that miR-21 is released into EVs and is neurotoxic when compared to EVs derived from miR-21-/-knockout animals. MiR-21 alone, incorporated into EVs, is neurotoxic, and a mutation of the sequence within miR-21, predicted to bind TLR7, eliminates this neurotoxicity. Indeed miR-21 in EV activates TLR7 in a reporter cell line, and the neurotoxicity is dependent upon TLR7, as neurons isolated from TLR7-/-knockout mice are protected from neurotoxicity. We also found that EVs isolated from the brains of monkeys with SIV induced CNS disease activate TLR7 and were neurotoxic when compared to EVs from control animals. Finally, we show that EV-miR-21 induced neurotoxicity could be prevented by a necroptosis inhibitor, highlighting the actions of this pathway in a growing number of CNS disorders. The ability to isolate and analyze, molecularly and functionally, EVs from tissues such as the brain enables the study of their role in HIV pathogenesis. This work is supported by MH106422 and MH062261.