Jonas A. Blöcher, Marleen J. Meyer‐Tönnies, Felix Morof, Vincent Rönnpagel, Jonas Bethmann, Marcus Vollmer, Stefan Engeli, Mladen V. Tzvetkov

• An over‐the‐counter product berberine (a major alkaloid in goldenseal) is a substrate of the uptake transporter OCT1 and the metabolizing enzyme CYP2D6. The two genes exhibit common functional polymorphisms. Approximately 9% of Europeans and white Americans are either poor CYP2D6 metabolizers or poor OCT1 transporters. In this study, we investigated the effects of OCT1 and CYP2D6 polymorphisms on berberine pharmacokinetics in humans. We confirmed in vitro that berberine is an OCT1 substrate (KM of 7.0 μM, CLint of 306 ± 29 μL/min/mg). Common OCT1 alleles *3 to *6 showed uptake reduced by at least 65% and Oct1/2 knockout mice showed 3.2‐fold higher AUCs in liver perfusion experiments. However, in humans, poor OCT1 transporters did not show any differences in berberine pharmacokinetics compared with reference participants. In contrast, CYP2D6 polymorphisms significantly affected berberine metabolism, but exclusively in females. Females who were poor CYP2D6 metabolizers had an 80% lower M1‐to‐berberine ratio. General linear model analyses suggest strong synergistic, rather than additive, effects between female sex and CYP2D6 genotype. Overall, berberine displayed low oral bioavailability, yet females had a 2.8‐fold higher AUC and a 3.6‐fold higher Cmax than males (P < 0.001). These effects were only partially attributable to the sex‐CYP2D6 genotype interaction. In conclusion, despite berberine being an OCT1 substrate, OCT1 deficiency did not affect berberine pharmacokinetics in humans. In contrast, CYP2D6 emerges as a critical enzyme for berberine metabolism in females, but not in males, highlighting sex‐specific differences. We suggest that factors beyond CYP2D6 metabolism are determining berberine's systemic exposure, especially in males (NCT05463003).