OBJECTIVE:: Viral infections influence intracellular peptide repertoires available for presentation by HLA-I. Alterations in HLA-I/peptide complexes can modulate binding of KIRs and thereby the function of NK cells. While multiple studies have provided evidence that HLA-I/KIR interactions play a role in HIV-1 disease progression, the consequence of HIV-1 infection for HLA-I/KIR interactions remain largely unknown. DESIGN:: We determined changes in HLA-I-presented peptides resulting from HIV-1 infection of primary human CD4 T-cells and assessed the impact of changes in peptide repertoires on HLA-I/KIR interactions. METHODS:: Liquid chromatography-coupled tandem mass spectrometry to identify HLA-I presented peptides, cell-based in vitro assays to evaluate functional consequences of alterations in immunopeptidome and atomistic molecular dynamics simulations to confirm experimental data. RESULTS:: A total of 583 peptides exclusively presented on HIV-1-infected cells were identified, of which only 1.4% represented HIV-1-derived peptides. Focusing on HLA-C*03:04/KIR2DL3 interactions, we observed that HLA-C*03:04-presented peptides derived from non-infected CD4 T-cells mediated stronger binding of inhibitory KIR2DL3 than peptides derived from HIV-1-infected cells. Furthermore, the most abundant peptide presented by HLA-C*03:04 on non-infected CD4 T-cells (VIYPARISL) mediated the strongest KIR2DL3-binding, while the most abundant peptide presented on HIV-1-infected cells (YAIQATETL) did not mediate KIR2DL3-binding. Molecular dynamics simulations of HLA-C*03:04/KIR2DL3 interactions in the context of these two peptides revealed that VIYPARISL significantly enhanced the HLA-C*03:04/peptide contact area to KIR2DL3 compared to YAIQATETL. CONCLUSIONS:: These data demonstrate that HIV-1 infection-induced changes in HLA-I-presented peptides can reduce engagement of inhibitory KIRs, providing a mechanism for enhanced activation of NK cells by virus-infected cells.