Mitragyna speciosa (Kratom) has emerged as a recreational drug and a substance of medicinal intrigue. Although the drug was initially used recreationally for its sedating and euphoric effects, more recently its use has been associated with the non-medically supervised treatment of opioid abstinence syndrome. Mitragynine is the principal pharmacologically active alkaloid in kratom. Although metabolites of mitragynine have been identified, the cytochrome P450 (CYP450) enzymes responsible for its biotransformation are still under investigation. The goal of this study was to contribute further knowledge regarding CYP450 activity as it relates to mitragynine. Recombinant cytochrome P450 enzymes (rCYPs) were used to investigate the isoforms involved in its metabolism. Biotransformational products were identified using liquid chromatography-quadrupole/time of flight-mass spectrometry. Four rCYP enzymes (2C18, 2C19, 2D6 and 3A4) were found to contribute to the metabolism of mitragynine. 7-Hydroxymitragynine (which has an affinity for the mu-opioid receptor >10-folds that of morphine) was produced exclusively by 3A4. 9-O-demethylmitragynine, the most abundant metabolite in vitro (and the most prevalent metabolite in urine among kratom users) was produced by 2C19, 3A4 and 2D6. 16-Carboxymitragynine was produced by rCYPs 2D6, 2C19 and 2C18. 2C19 was solely responsible for the formation of 9-O-demethyl-16-carboxymitragynine. In vitro rCYP studies were compared with phase I metabolites in urine from cases involving mitragynine.