NKG2D (natural killer group 2, member D) is an activating receptor found on the surface of immune cells, including natural killer (NK) cells, which regulates innate and adaptive immunity through recognition of the stress-induced ligands ULBP1 (UL16 binding protein 1) to ULBP6 and MICA/B. Similar to class I human leukocyte antigen (HLA), these NKG2D ligands have a major histocompatibility complex–like fold and exhibit pronounced polymorphism, which influences human disease susceptibility. However, whereas class I HLA polymorphisms occur predominantly in the α1α2 groove and affect antigen binding, the effects of most NKG2D ligand polymorphisms are unclear. We studied the molecular and functional consequences of the two major alleles of ULBP6, the most polymorphic ULBP gene, which are associated with autoimmunity and relapse after stem cell transplantation. Surface plasmon resonance and crystallography studies revealed that the arginine-to-leucine polymorphism within ULBP0602 affected the NKG2D-ULBP6 interaction by generating an energetic hotspot. This resulted in an NKG2D-ULBP0602 affinity of 15.5 nM, which is 10- to 1000-fold greater than the affinities of other ULBP-NKG2D interactions and limited NKG2D-mediated activation. In addition, soluble ULBP0602 exhibited high-affinity competitive binding for NKG2D and partially suppressed NKG2D-mediated activation of NK cells by other NKG2D ligands. These effects resulted in a decrease in a range of NKG2D-mediated effector functions. Our results reveal that ULBP polymorphisms affect the strength of human lymphocyte responses to cellular stress signals and may offer opportunities for therapeutic intervention.