The plasma membrane proteins CD1a, CD1b and CD1c are expressed by human dendritic cells, the professional antigen‐presenting cells of the immune system, and present lipid antigens to T lymphocytes. CD1e belongs to the same family of molecules, but accumulates as a membrane‐associated form in the Golgi compartments of immature dendritic cells and as a soluble cleaved form in the lysosomes of mature dendritic cells. In lysosomes, the N‐terminal propeptide of CD1e is also cleaved, but the functional consequences of this step are unknown. Here, we investigated how the pH changes encountered during transport to lysosomes affect the structure of CD1e and its ligand‐binding properties. Circular dichroism studies demonstrated that the secondary and tertiary structures of recombinant CD1e were barely altered by pH changes. Nevertheless, at acidic pH, guanidium chloride‐induced unfolding of CD1e molecules required lower concentrations of denaturing agent. The nonfunctional L194P allelic variant was found to be structurally less stable at acidic pH than the functional forms, providing an explanation for the lack of its detection in lysosomes. The number of water‐exposed hydrophobic patches that bind 8‐anilinonaphthalene‐1‐sulfonate was higher in acidic conditions, especially for the L194P variant. CD1e molecules interacted with lipid surfaces enriched in anionic lipids, such as bis(monoacylglycero)phosphate, a late endosomal/lysosomal lipid, especially at acidic pH, or when the propeptide was present. Altogether, these data indicate that, in the late endosomes/lysosomes of DCs, the acid pH promotes the binding of lipid antigens to CD1e through increased hydrophobic and ionic interactions.