BACKGROUND AND PURPOSE Mass drug administration of ivermectin has been proposed as a possible malaria elimination tool. Ivermectin exhibits a mosquito-lethal effect well beyond its biological half-life, suggesting the presence of active slowly eliminated metabolites. EXPERIMENTAL APPROACH Human liver microsomes, primary human hepatocytes, and whole blood from healthy volunteers given oral ivermectin were used to identify ivermectin metabolites by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry. The molecular structures of metabolites were determined by mass spectrometry and verified by nuclear magnetic resonance. Pure cytochrome P450 enzyme isoforms were used to elucidate the metabolic pathways. KEY RESULTS Thirteen different metabolites (M1-M13) were identified after incubation of ivermectin with human liver microsomes. Three (M1, M3, and M6) were the dominant metabolites found in microsomes, hepatocytes, and blood from volunteers after oral ivermectin administration. The chemical structure defined by LC-MS/MS and NMR indicated that M1 is 3″-O-demethyl ivermectin, M3 is 4-hydroxymethyl ivermectin, and M6 is 3″-O-demethyl, 4-hydroxymethyl ivermectin. Metabolic pathway evaluations with characterized cytochrome P450 enzymes showed that M1 was produced by CYP3A4 and CYP3A5, and that M3 and M6 were produced by CYP3A4. CONCLUSIONS AND IMPLICATIONS Demethylated and hydroxylated ivermectin are the main human metabolites in vivo. Further study to characterize their pharmacokinetic properties and mosquito-lethal activity is now needed.