Mycotoxins are secondary metabolites of filamentous fungi which can cause a wide range of systemic effects. Humanhealth effects of inhaled mycotoxins remain poorly documented, despite the large amounts present, associated with airborneparticles.Among thesemycotoxins, sterigmatocystin is one of themost prevalent. Because its chemical structure isclose to that of the aflatoxins, we studied its metabolism and its cellular consequences when in contact with the airwayepithelium, using the mass spectral signature from the 10% 13C uniformly enriched sterigmatocystin. The metabolismwas studied in vitro, using recombinant cytochrome P450s enzymes, and in porcine tracheal epithelial cell (PTEC)primary cultures at an air‐liquid interface. Themetabolites were analyzed by high‐performance liquid chromatographycoupled with tandem mass spectrometry detection. Expressed enzymes and PTECs were exposed to uniformly 13Cenrichedsterigmatocystin to confirm the relationship between sterigmatocystin and its metabolites because this isotopiccluster shape is conserved for all metabolites and their product ions. Incubation of sterigmatocystin with recombinantcytochrome P450 1A1 led to the formation of three metabolites identified as monohydroxysterigmatocystin,dihydroxysterigmatocystin and one glutathione adduct, the latter after the formation of a transient intermediate. Inthe PTEC cultures, sterigmatocystin metabolism resulted in a glucuro‐conjugate. Two other products were detected, asulfo‐conjugate and a glucuro‐conjugate of hydroxysterigmatocystin upon cytochrome P450 1A1 induction. This is thefirst study to report sterigmatocystin metabolism in airway epithelium, and it suggests that, contrary to the aflatoxins,sterigmatocystin is mainly detoxified into its conjugates and is unable to produce significant amounts of reactivemetabolites in respiratory cells, at least in pigs.