Significance Invariant natural killer T (iNKT) cells are activated quickly and play a key role in the control of many microbial infections via their ability to rapidly secrete cytokines and chemokines that enhance many immune responses. Microbial glycolipid antigens that activate iNKT cells have been identified as α-anomerically linked glycolipids. However, the nature of the endogenous lipid antigens that are important contributors to the biology of iNKT cells has been unclear. In this study, antigenic lipids from cow’s milk were isolated using a T-cell receptor trap method, and their stereochemical structures were determined to be hexosylceramides with α-linked hexose headgroups, thus identifying dietary lipid antigens for iNKT cells.