Aspergillus fumigatus is a saprophytic fungus that is ubiquitous in the environment and is commonly associated with allergic sensitization and severe asthma in humans. Although A. fumigatus is recognized by multiple microbial pattern-recognition receptors, we found that an A. fumigatus–derived glycosphingolipid, asperamide B, directly activates invariant natural killer T (iNKT) cells in vitro in a CD1d-restricted, MyD88-independent and dectin-1–independent fashion. Moreover, asperamide B, when loaded onto CD1d, directly stained, and was sufficient to activate, human and mouse iNKT cells. In vivo, asperamide B rapidly induced airway hyperreactivity, which is a cardinal feature of asthma, by activating pulmonary iNKT cells in an interleukin-33 (IL-33)-ST2–dependent fashion. Asperamide B is thus the first fungal glycolipid found to directly activate iNKT cells. These results extend the range of microorganisms that can be directly detected by iNKT cells to the kingdom of fungi and may explain how A. fumigatus can induce severe chronic respiratory diseases in humans.