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.