KIR3DL1 is a polymorphic inhibitory receptor that modulates NK cell activity through interacting with HLA-A and HLA-B alleles that carry the Bw4 epitope. Amino acid polymorphisms throughout KIR3DL1 impact receptor surface expression and affinity for HLA. KIR3DL1/S1 encodes inhibitory and activating alleles, but despite high homology with KIR3DL1, the activating receptor KIR3DS1 does not bind the same ligand. Allele KIR3DL1009 resulted from a gene recombination event between the inhibitory receptor allele KIR3DL1001 and the activating receptor allele KIR3DS1013. This study analyzed the functional impact of KIR3DS1-specific polymorphisms on KIR3DL1009 surface expression, binding to HLA, and functional capacity. Flow-cytometric analysis of primary human NK cells as well as transfected HEK293T cells shows that KIR3DL1009 is expressed at a significantly lower surface density compared with KIR3DL1001. Using recombinant proteins of KIR3DL1001, KIR3DL1009, and KIR3DS1013 to analyze binding to HLA, we found that although KIR3DL1009 displayed some evidence of binding to HLA compared with KIR3DS1013, the binding was minimal compared with KIR3DL1001 and KIR3DL1005. Mutagenesis of polymorphic sites revealed that the surface phenotype and reduced binding of KIR3DL1009 are caused by the combined amino acid polymorphisms at positions 58 and 92 within the D0 extracellular domain. Resulting from these effects, KIR3DL1009(+) NK cells exhibited significantly less inhibition by HLA-Bw4(+) target cells compared with KIR3DL1001(+) NK cells. The data from this study contribute novel insight into how KIR3DS1-specific polymorphisms in the extracellular region impact KIR3DL1 surface expression, ligand binding, and inhibitory function.