To inhibit endocytic entry of some viruses, cells promote acidification of endosomes by expressing the short isoform of human nuclear receptor 7 (NCOA7) which increases activity of vacuolar ATPase (V-ATPase). While we found that HIV-1 infection of primary T cells led to acidification of endosomes, NCOA7 levels were only marginally affected. Contrastingly, levels of the 50 kDa form of the sodium/hydrogen exchanger 6 (NHE6) were greatly reduced. NHE6 overexpression and low concentrations of the V-ATPase inhibitor, concanamycin A, selectively reversed endosomal acidification. Endosomal neutralization by these interventions also reduced Nef-dependent MHC-I downmodulation by our wildtype HIV reporter virus. NHE6 overexpression disrupted MHC-I downmodulation by reducing recruitment of Nef to Rab11 + compartments and inhibiting interactions between Nef, β-COP, and ARF-1. In addition, we found that the HIV Vif protein was required for downmodulation of the 50 kDa form of NHE6 and for endosomal acidification but was dispensable for Nef-dependent MHC-I downmodulation.