Human and mouse type I NKT cells respond to a variety of CD1d-restricted glycolipid antigens (Ags), with their NKT cell antigen receptors (NKT TCRs) exhibiting reciprocal cross-species reactivity that is underpinned by a conserved NKT TCR-CD1d-Ag docking mode. Within this common docking footprint, the NKT TCR recognises, to varying degrees of affinity, a range of Ags. Presently it is unclear whether the human NKT TCRs will mirror the generalities underpinning the fine specificity of the mouse NKT TCR-CD1d-Ag interaction. Here, we assessed human NKT TCR recognition against altered glycolipid ligands of α-galactosylceramide (α-GalCer), and have determined the structures of a human NKT TCR in complex with CD1d-4',4''-deoxy-α-GalCer and CD1d-α-GalCer with a shorter, di-unsaturated acyl chain (C20:2). AGLs with acyl-chain modifications did not affect the affinity of the human NKT TCR-CD1d-Ag interaction. Surprisingly, human NKT TCR recognition is more tolerant to modifications at the 4'-OH position in comparison to the 3'-OH position of α-GalCer, which contrasts the fine specificity of the mouse NKT TCR-CD1d-Ag recognition (4'-OH > 3'-OH). The fine specificity differences between human and mouse NKT TCRs was attributable to differing interactions between the respective complementarity determining region (CDR) 1α loops and the Ag. Accordingly, germline encoded fine-specificity differences underpin human and mouse type I NKT TCR interactions, which is an important considerations for therapeutic development and NKT cell physiology.