Little is known regarding the basis for selection of the semi-invariant αβ T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d–glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3β composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR α- and semi-invariant β-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved “hot spot” that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3β loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR–CD1d–glycolipid interaction in which the invariant CDR3α loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant αβ TCR and the unique antigen specificity of NKT cells.