Although Nef is the viral gene product used by most simian immunodeficiency viruses to overcome restriction by tetherin, this activity was acquired by the Vpu protein of HIV-1 groupMdue to the absence of sequences in human tetherin that confer susceptibility to Nef. Thus, it is widely accepted that HIV-1 groupMuses Vpu instead of Nef to counteract tetherin. Challenging this paradigm, we identified Nef alleles of HIV-1 groupMisolates with significant activity against human tetherin. These Nef proteins promoted virus release and tetherin downmodulation from the cell surface and, in the context of vpu-deleted HIV-1 recombinants, enhanced virus replication and resistance to antibody-dependent cell-mediated cytotoxicity (ADCC). Further analysis revealed that the Vpu proteins from several of these viruses lack antitetherin activity, suggesting that under certain circumstances, HIV-1 groupMNef may acquire the ability to counteract tetherin to compensate for the loss of this function by Vpu. These observations illustrate the remarkable plasticity of HIV-1 in overcoming restriction by tetherin and challenge the prevailing view that all HIV-1 groupMisolates use Vpu to counteract tetherin.