Although antiretroviral therapy is highly effective in suppressing human immunodeficiency virus type-1 (HIV) replication, treatment has failed to eliminate viral reservoirs and discontinuation of treatment results in viral reactivation. Here, we demonstrate that peptides Tat-vFLIP-α2 and Tat-Beclin 1/BECN1 which have been shown to induce a Na⁺/K⁺-ATPase- and a macroautophagy/autophagy-dependent form of cell death, autosis, can preferentially kill HIV-infected macrophages while preventing virological rebound. To improve bioavailability and drug delivery, Tat-vFLIP-α2 was encapsulated into biodegradable PLGA (poly lactic-co-glycolic acid)-lipid-PEG (polyethylene glycol) nanoparticles for long-lasting intracellular delivery. After a single dose of NP-vFLIP-α2, HIV-infected macrophages were preferentially killed in a dose-dependent manner compared to uninfected or untreated HIV-infected cells with complete inhibition of HIV infection at 10 μM of peptide. HIV-infected macrophages treated with NP-vFLIP-α2 exhibited increased markers of autophagy including LC3B lipidation, SQSTM1/p62 degradation and Na⁺/K⁺-ATPase expression compared to untreated uninfected or infected cells. Moreover, the increased cell death observed in HIV-infected cells was not altered by treatment with bafilomycin A₁ (BAF) or the caspase inhibitor Z-VAD-FMK, but could be reversed following treatment with the Na⁺/K⁺-ATPase inhibitor, digoxin, or knockdown of ATG5 or ATG7. NP-vFLIP-α2 induced preferential killing was also detected in HIV-infected macrophages under antiretroviral suppression without inducing viral reactivation. Additionally, we found that Na⁺/K⁺-ATPase was upregulated in HIV-infected cells, which enhanced NP-vFLIP-α2 induced cell death. These findings provide a novel strategy to eradicate HIV-infected macrophages by selectively killing infected cells through the induction of Na⁺/K⁺-ATPase dependent autophagy, while preventing reactivation of virus and new infection of uninfected bystander cells.