NKT cells are a unique subset of T cells that express a conserved TCR and NK receptors. While their TCR is typically autoreactive to CD1d, a lipid-presenting molecule, the endogenous ligands for these cells have remained elusive. This study identifies isoglobotrihexosylceramide (iGb3), a lysosomal glycosphingolipid, as a natural ligand for both mouse and human NKT cells. Mice deficient in β-hexosaminidase b (Hexb) show severe NKT cell deficiency, suggesting that iGb3 is essential for NKT cell development. iGb3 is expressed in peripheral tissues and may regulate NKT cell responses to infections, malignancy, and autoimmunity. The study demonstrates that iGb3 is processed in the lysosome by β-hexosaminidase b to generate a form recognized by NKT cells. iGb3 selectively expands human Vα24 NKT cells and stimulates Th1 and Th2 cytokine secretion. It is presented by CD1d-expressing dendritic cells and is recognized by both mouse and human NKT cells. The study also shows that iGb3 is processed in the lysosome by β-hexosaminidase b to generate a form recognized by NKT cells. The binding of iGb3 to CD1d requires saposin B, and the recognition of iGb3 by NKT cells is specific to its terminal Gal α1,3 Gal residues. The findings suggest that iGb3 is the principal endogenous ligand for NKT cells, and its expression is essential for NKT cell development. The study also highlights the importance of lysosomal processing in the presentation of iGb3 to NKT cells. The results indicate that iGb3 or a close structural analog is the principal self antigen of NKT cells. The study also suggests that additional endogenous ligands may exist, and that the dysregulation of iGb3 expression may contribute to diseases regulated by NKT cells. The discovery of natural endogenous and microbial NKT cell ligands, and the synthesis of pharmacologic agonists or inhibitors, may lead to novel approaches to manipulating NKT cells for the prevention and treatment of diseases.NKT cells are a unique subset of T cells that express a conserved TCR and NK receptors. While their TCR is typically autoreactive to CD1d, a lipid-presenting molecule, the endogenous ligands for these cells have remained elusive. This study identifies isoglobotrihexosylceramide (iGb3), a lysosomal glycosphingolipid, as a natural ligand for both mouse and human NKT cells. Mice deficient in β-hexosaminidase b (Hexb) show severe NKT cell deficiency, suggesting that iGb3 is essential for NKT cell development. iGb3 is expressed in peripheral tissues and may regulate NKT cell responses to infections, malignancy, and autoimmunity. The study demonstrates that iGb3 is processed in the lysosome by β-hexosaminidase b to generate a form recognized by NKT cells. iGb3 selectively expands human Vα24 NKT cells and stimulates Th1 and Th2 cytokine secretion. It is presented by CD1d-expressing dendritic cells and is recognized by both mouse and human NKT cells. The study also shows that iGb3 is processed in the lysosome by β-hexosaminidase b to generate a form recognized by NKT cells. The binding of iGb3 to CD1d requires saposin B, and the recognition of iGb3 by NKT cells is specific to its terminal Gal α1,3 Gal residues. The findings suggest that iGb3 is the principal endogenous ligand for NKT cells, and its expression is essential for NKT cell development. The study also highlights the importance of lysosomal processing in the presentation of iGb3 to NKT cells. The results indicate that iGb3 or a close structural analog is the principal self antigen of NKT cells. The study also suggests that additional endogenous ligands may exist, and that the dysregulation of iGb3 expression may contribute to diseases regulated by NKT cells. The discovery of natural endogenous and microbial NKT cell ligands, and the synthesis of pharmacologic agonists or inhibitors, may lead to novel approaches to manipulating NKT cells for the prevention and treatment of diseases.