Combinations of KIR3DL1 and HLA-Bw4 alleles protect against HIV infection and/or disease progression. These combinations enhance NK cell responsiveness through the ontological process of education. However, educated KIR3DL1(+) NK cells do not have enhanced degranulation upon direct recognition of autologous HIV-infected cells. Since antibody-dependent cellular cytotoxicity (ADCC) is associated with improved HIV infection outcomes and NK cells overcome inhibition through killer cell immunoglobulin-like receptors (KIR) to mediate ADCC, we hypothesized that KIR3DL1-educated NK cells mediate anti-HIV ADCC against autologous cells. A whole-blood flow cytometry assay was used to evaluate ADCC-induced activation of NK cells. This assay assessed activation (gamma interferon [IFN-γ] production and/or CD107a expression) of KIR3DL1(+) and KIR3DL1(-) NK cells, from HLA-Bw4(+) and HLA-Bw4(-) HIV-positive and HIV-negative individuals, in response to autologous HIV-specific ADCC targets. KIR3DL1(+) NK cells were more functional than KIR3DL1(-) NK cells from HLA-Bw4(+), but not HLA-Bw4(-), healthy controls. In HIV-infected individuals, no differences in NK cell functionality were observed between KIR3DL1(+) and KIR3DL1(-) NK cells in HLA-Bw4(+) individuals, consistent with dysfunction of NK cells in the setting of HIV infection. Reflecting the partial normalization of NK cell responsiveness following initiation of antiretroviral therapy, a significant correlation was observed between the peripheral CD4(+) T-lymphocyte counts in antiretroviral therapy-treated subjects and the functionality of NK cells. However, peripheral CD4(+) T-lymphocyte counts were not correlated with an anti-HIV ADCC functional advantage in educated KIR3DL1(+) NK cells. The abrogation of the functional advantage of educated NK cells may enhance HIV disease progression. Strategies to enhance the potency of NK cell-mediated ADCC may improve HIV therapies and vaccines.