Killer cell immunoglobulin-like receptors (KIRs) and their HLA ligands interact to regulate natural killer (NK) cell function. KIR gene content and allelic variations are reported to influence human immunodeficiency virus (HIV)-1 infection and pathogenesis. We investigated the impact of KIR genes on heterosexual HIV-1 transmission among 566 discordant couples from Lusaka, Zambia. KIR2DS4*001, the only allele of KIR2DS4 known to encode a functional activating receptor, was associated with relatively high viral load for HIV-1 in index (HIV-1 seroprevalent) partners (β [standard error (SE)], .17 [.8] log10; P = .04) and with accelerated transmission of HIV-1 to cohabiting seronegative partners (relative hazard [RH], 2.00; P = .004). The latter association was independent of the direction of transmission (male-to-female or female-to-male), genital ulcers, and carriage of the putative ligand (HLA-Cw*04). No KIR-gene variant in the initially seronegative partners was associated with HIV-1 acquisition or early viral load following seroconversion. Further analysis of NK cell function should clarify the role of KIR2DS4*001 in HIV-1 transmission. © The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved.