The viral accessory protein Nef is a major determinant of HIV-1 pathogenicity in vivo. Nef is a multifunctional, immunomodulatory protein that downmodulates cell surface proteins, including CD4 and MHC class I (MHC-I), which are important for T cell-mediated immunity. In addition, Nef also regulates cell-intrinsic immunity-Nef boosts the infectivity of virions produced and released from HIV-infected cells, at least in part, by counteracting the antiviral activity of transmembrane proteins SERINC3 and SERINC5. Here, we show that Nef proteins derived from many primary isolates of HIV-1 restore infectivity in interferon-treated cells and confer resistance to the antiviral protein interferon-induced transmembrane protein 3 (IFITM3) in a SERINC3/5-independent manner. Using Nef derived from primary HIV-1 clade C infection, we found that Nef interacts with IFITM3 in membranes, reduces IFITM3 incorporation into HIV-1 virions, and restores HIV-1 fusion with target cells. Our findings reveal a previously unrecognized immunomodulatory role for Nef in the setting of the interferon-induced antiviral state during HIV-1 infection.