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Human Leukocyte Antigen (HLA)-G is selectively expressed by fetal trophoblast cells that encounter maternal natural killer (NK) cells early in pregnancy. In response to HLA-G, the endosomal NK cell receptor KIR2DL4 induces a transcriptional program to support placental development. Structural features of KIR2DL4 that control HLA-G binding and endocytosis are unclear. Random mutagenesis revealed that three cysteines in the first immunoglobulin domain of KIR2DL4 regulate endocytosis and uptake of HLA-G. The atypical Cys10-Cys28 disulfide bond in the KIR2DL4 structure is an allosteric disulfide with potential to switch to a conventional Cys28-Cys74 bond. KIR2DL4 in human cells exists in both disulfide-bonded states, as shown by mass spectrometry analysis. The Cys10-Cys28 bond in a Cys74Ser KIR2DL4 mutant was reduced by protein disulfide isomerase (PDI) in vitro. Inhibition of PDI prevented HLA-G uptake by KIR2DL4 in NK cells. Mutants in the Cys10-Cys28 configuration endocytosed spontaneously but did not bind HLA-G. Conversely, KIR2DL4 with a Cys28-Cys74 bond was detectable at the plasma membrane and internalized soluble HLA-G. A purified Cys10Leu KIR2DL4 mutant bound HLA-G with micromolar affinity in a peptide-dependent manner. Thus, allosteric disulfide-mediated conversion from an inactive to an HLA-G binding form regulates KIR2DL4 trafficking and a functional response that promotes fetal development.