Eltrombopag (ELT) is a novel thrombopoietin receptor agonist for the treatment of idiopathic thrombocytopenic purpura. Previous reports indicate that ELT is mainly eliminated in the liver, although its pharmacokinetic profile has not yet been clarified in detail. The purpose of the present study is to investigate the overall elimination mechanism of ELT. After intravenous administration of ELT to rats, approximately 40% of unchanged ELT was excreted into the bile in 72 h, whereas less than 0.02% of the dose was excreted in urine, indicating that liver is the major elimination organ for ELT. The total clearance was much lower than the hepatic blood flow rate and comparable with hepatic uptake clearance obtained from integration plot analysis. Coadministration of rifampicin, an organic anion transporter inhibitor, reduced both total clearance and hepatic uptake clearance of ELT. These results suggest that hepatic uptake is the rate-limiting process in the overall elimination of ELT. To further characterize the uptake mechanism, uptake of ELT by freshly isolated mouse hepatocytes was examined. The ELT uptake showed concentration and energy dependence and was inhibited by various compounds, including not only organic anions but also organic cations. Hepatic uptake clearance in vivo was reduced by coadministration of an organic cation, tetrapentylammonium. Finally, uptake of ELT was observed in human embryonic kidney 293 cells transfected with human hepatic transporters organic anion-transporting polypeptide (OATP) 1B1 and OATP2B1 and organic cation transporter OCT1. These results suggest that multiple transporters, including organic anion transporters and organic cation transporters, are involved in hepatic ELT uptake.