Eudesmin is a tetrahydrofurofuranoid lignan with various pharmacological activities, including anti-tumor and neuroprotective effects. This study characterized the in vitro metabolism of eudesmin using human and mouse hepatocytes, human liver microsomes, and recombinant enzymes. To comprehensively visualize the metabolic network, liquid chromatography–high-resolution mass spectrometry was integrated with ion identity molecular networking. Incubation of eudesmin with human hepatocytes resulted in the formation of five phase I [O-desmethyleudesmin (M1 and M2), di-O-desmethyleudesmin (M3 and M4), and hydroxyeudesmin (M5)] and eight phase II conjugates (glucuronides of M1–M4 and sulfates of M1–M3), whereas 3 phase I (M1, M2, and M5) and 4 phase II metabolites (glucuronides and sulfates of M1 and M2) were identified in mouse hepatocytes. M3 was identified as pinoresinol, a bioactive lignan with antioxidant and anti-inflammatory activity. Enzyme phenotyping and chemical inhibition experiments showed that cytochrome P450 2C9 (CYP2C9) played a major role in eudesmin O-demethylation to M1–M4, whereas CYP3A4 and CYP3A5 were primarily responsible for hydroxylation to M5. Glucuronidation and sulfation of M1 and M3 were catalyzed by multiple uridine 5′-diphospho-glucuronosyltransferase and sulfotransferase isoforms. Eudesmin was moderately metabolized via O-demethylation, hydroxylation, glucuronidation, and sulfation in human and mouse hepatocytes.