Murine NKT cells can recognize α-galactosylceramide (α-GalCer) in the context of a class Ib CD1d molecule. Here we show that α-GalCer can selectively activate freshly isolated human V_α24⁺V_β11⁺ cells, functionally defining the human NKT cells. The naive human NKT cell repertoire consisted of cells expressing an invariant V_α24J_αQ chain and a diverse array of β chains derived from a single V_β11 gene segment. Stimulation with α-GalCer expanded a polyclonal subset of the human NKT cell repertoire carrying a novel complementarity-determining region (CDR) 3β consensus motif that may directly interact with the sugar moiety of α-GalCer. Our data suggest that certain redundancy is allowed for CDR3β of NKT antigen receptor to interact with the ligand and provide a first clue to understand the novel protein–carbohydrate interaction mechanisms.