The non-classical major histocompatibility complex (MHC) class I molecule CD1d presents lipid antigens to invariant natural killer T (iNKT) cells, which are an important part of the innate immune system. CD1d/iNKT systems are highly conserved in evolution, and cross-species reactivity has been suggested to be a common feature of different animals based on research in humans and mice. However, we found that CD1d from the tree shrew (Tupaia belangeri), a close evolutionary relative of primates, failed to stimulate human iNKT cells, despite being more homologous to human CD1d than that of mouse. Sequence comparison and molecular modelling showed that two of the key amino acid residues in human CD1d proposed to be in direct contact with T-cell receptors were mutated in tree shrew CD1d. Substitution of one of the residues, but not the other, with the human residue enabled tree shrew CD1d to regain the ability to present lipid antigen to human iNKT cells. These results indicate that CD1d/iNKT recognition is species-specific, and that cross-species reactivity may be less common than currently proposed. Also, a naturally occurring CD1d mutation(s) that confers inability to stimulate iNKT cell function may have implications for future studies on CD1d/iNKT-associated diseases.