R (−)-Apomorphine is a dopamine agonist used for rescue management of motor function impairment associated with levodopa therapy in Parkinson’s disease patients. The aim of this study was to examine the role of proton-coupled organic cation antiporter in uptake of R (−)-apomorphine and its S -enantiomer in human brain, using human endothelial cell line hCMEC/D3 as a model. Uptake of R (−)- or S (+)-apomorphine into hCMEC/D3 cells was measured under various conditions to evaluate its time-, concentration-, energy- and ion-dependency. Inhibition by selected organic cations was also examined. Uptakes of both R (−)- and S (+)-apomorphine increased with time. The initial uptake velocities of R (−)- and S (+)-apomorphine were concentration-dependent, with similar K _m and V _max values. The cell-to-medium (C/M) ratio of R (−)-apomorphine was significantly reduced by pretreatment with sodium azide, but was not affected by replacement of extracellular sodium ion with N -methylglucamine or potassium. Intracellular alkalization markedly reduced the uptake, while intracellular acidification increased it, suggesting that the uptake is driven by an oppositely directed proton gradient. The C/M ratio was significantly decreased by amantadine, verapamil, pyrilamine and diphenhydramine (substrates or inhibitors of proton-coupled organic cation antiporter), while tetraethylammonium (substrate of organic cation transporters (OCTs)) and carnitine (substrate of carnitine/organic cation transporter 2; (OCTN2)) had no effect. R (−)-Apomorphine uptake was competitively inhibited by diphenhydramine. Our results indicate that R (−)-apomorphine transport in human blood–brain barrier (BBB) model cells is similar to S (+)-apomorphine uptake. The transport was dependent on an oppositely directed proton gradient, but was sodium- or membrane potential-independent. The transport characteristics were consistent with involvement of the previously reported proton-coupled organic cation antiporter.