Understanding the mechanisms of drug transport across the blood-brain barrier (BBB) is an important issue for regulating the pharmacokinetics of drugs in the central nervous system. In this study, we focused on solute carrier family 35, member F2 (SLC35F2), whose mRNA is highly expressed in the BBB. SLC35F2 protein was enriched in isolated mouse and monkey brain capillaries relative to brain homogenates and was localized exclusively on the apical membrane of MDCKII cells and brain microvascular endothelial cells (BMECs) differentiated from human induced pluripotent stem cells (hiPS-BMECs). SLC35F2 activity was assessed using its substrate, YM155, and pharmacological experiments revealed SLC35F2 inhibitors, such as famotidine (half-maximal inhibitory concentration, 160 μM). Uptake of YM155 was decreased by famotidine or SLC35F2 siRNA transfection in immortalized human BMECs (hCMEC/D3 cells). Furthermore, famotidine significantly inhibited the apical-to-basal (A-to-B) transport of YM155 in primary cultured monkey BMECs and hiPS-BMECs. Crucially, SLC35F2-knockout diminished the A-to-B transport and intracellular accumulation of YM155 in hiPS-BMECs. By contrast, in studies using an in situ brain perfusion technique, neither deletion of Slc35f2 nor famotidine reduced brain uptake of YM155, even though YM155 is a substrate of mouse SLC35F2. YM155 uptake was decreased significantly by losartan and naringin, inhibitors for the organic anion transporting polypeptide 1A4 (OATP1A4). These findings suggest SLC35F2 is a functional transporter in various cellular models of the primate BBB that delivers its substrates to the brain and that its relative importance in the BBB is modified by differences in the expression of OATPs between primates and rodents. Significance Statement This study demonstrated that SLC35F2 is a functional drug influx transporter in three different cellular models of the primate blood-brain barrier (i.e., hCMEC/D3 cells, primary cultured monkey BMECs, and hiPS-BMECs), but has limited roles in mouse brain. SLC35F2 facilitates apical-to-basal transport across the tight cell monolayer. These findings will contribute to the development of improved strategies for targeting drugs to the central nervous system.