The immunomodulatory glycolipid α‐galactosylceramide (α‐GalCer) binds to CD1d and exhibits potent activity as a ligand for invariant CD1d‐restricted natural killer‐like T cells (iNKT cells). Structural analogues of α‐GalCer have been synthesised to determine which components are required for CD1d presentation and iNKT cell activation, however, to date the importance of the phytosphingosine 4‐hydroxyl for iNKT cell activation has been disputed. To clarify this, we synthesised two 4‐deoxy α‐GalCer analogues (sphinganine and sphingosine) and investigated their ability to activate murine and human iNKT cells. Analysis revealed that the analogues possessed comparable activity to α‐GalCer in stimulating murine iNKT cells, but were severely compromised in their ability to stimulate human iNKT cells. Here we determined that species‐specific glycolipid activity was due to a lack of recognition of the analogues by the T‐cell receptors on human iNKT cells rather than insufficient presentation of the analogues on human CD1d molecules. From these results we suggest that glycolipids developed for potent iNKT cell activity in humans should contain a phytosphingosine base.