Updated project metadata. Extracellular vesicles (EVs) are biomolecule carriers for intercellular communication in health and disease. Nef is a human immunodeficiency virus (HIV) virulence factor that is released from cells within EVs and is present in plasma EVs of HIV-1 infected individuals. We performed a quantitative proteomic analysis to fully characterize the Nef-induced changes in protein composition of T cell-derived EVs and identify novel host targets of HIV. Several proteins with well-described roles in infection or not previously associated with HIV pathogenesis were specifically modulated by Nef in EVs. Among the downregulated proteins are the interferon-induced transmembrane 1, 2 and 3 proteins (IFITM1-3), broad-spectrum antiviral factors known to be cell-to-cell transferable by EVs. Our data establish Nef as a modulator of EVs' global protein content and as an HIV factor that antagonizes IFITMs.