Isoglobotrihexosylceramide (iGb3) has been identified as a potent CD1d-presented self-antigen for mouse invariant natural killer T (iNKT) cells. The role of iGb3 in humans remains unresolved, however, as there have been conflicting reports about iGb3-dependent human iNKT-cell activation, and humans lack iGb3 synthase, a key enzyme for iGb3 synthesis. Given the importance of human immune responses, we conducted a human–mouse cross-species analysis of iNKT-cell activation by iGb3-CD1d. Here we show that human and mouse iNKT cells were both able to recognise iGb3 presented by mouse CD1d (mCD1d), but not human CD1d (hCD1d), as iGb3-hCD1d was unable to support cognate interactions with the iNKT-cell TCRs tested in this study. The structural basis for this discrepancy was identified as a single amino acid variation between hCD1d and mCD1d, a glycine-to-tryptophan modification within the α2-helix that prevents flattening of the iGb3 headgroup upon TCR ligation. Mutation of the human residue, Trp153, to the mouse ortholog, Gly155, therefore allowed iGb3-hCD1d to stimulate human iNKT cells. In conclusion, our data indicate that iGb3 is unlikely to be a major antigen in human iNKT-cell biology.