AbstractMitragyna speciosa is reported to be beneficial for the management of chronic pain and opioid withdrawal in the evolving opioid epidemic. Data on the blood–brain barrier (BBB) transport of mitragynine and 7‐hydroxymitragynine, the active compounds of the plant, are still lacking and inconclusive. Here, we present for the first time the rate and the extent of mitragynine and 7‐hydroxymitragynine transport across the BBB, with an investigation of their post‐BBB intra‐brain distribution. We utilized an in vitro BBB model to study the rate of BBB permeation of the compounds and their interaction with efflux transporter P‐glycoprotein (P‐gp). Mitragynine showed higher apical‐to‐basolateral (A‐B, i.e. blood‐to‐brain side) permeability than 7‐hydroxymitragynine. 7‐Hydroxymitragynine showed a tendency to efflux, with efflux ratio (B‐A/A‐B) of 1.39. Both were found to inhibit the P‐gp and are also subject to efflux by the P‐gp. Assessment of the extent of BBB transport in vivo in rats from unbound brain to plasma concentration ratios (Kp,uu,brain) revealed extensive efflux of both compounds, with less than 10 percent of unbound mitragynine and 7‐hydroxymitragynine in plasma crossing the BBB. By contrast, the extent of intra‐brain distribution was significantly different, with mitragynine having 18‐fold higher brain tissue uptake in brain slice assay compared with 7‐hydroxymitragynine. Mitragynine showed a moderate capacity to accumulate inside brain parenchymal cells, while 7‐hydroxymitragynine showed restricted cellular barrier transport. The presented findings from this systematic investigation of brain pharmacokinetics of mitragynine and 7‐hydroxymitragynine are essential for design and interpretation of in vivo experiments aiming to establish exposure–response relationship.