Valsartan is a highly selective angiotensin II AT1-receptor antagonist for the treatment of hypertension. Valsartan is mainly excreted into the bile in unchanged form. Because valsartan has an anionic carboxyl group, we hypothesized that a series of organic anion transporters could be involved in its hepatic clearance. In this study, to identify transporters that mediate the hepatic uptake and biliary excretion of valsartan and estimate the contribution of each transporter to the overall hepatic uptake and efflux, we characterized its transport using transporter-expressing systems, human cryopreserved hepatocytes, and Mrp2-deficient Eisai hyperbilirubinemic rats (EHBRs). Valsartan was significantly taken up into organic anion-transporting polypeptide (OATP) 1B1 (OATP2/OATP-C)- and OATP1B3 (OATP8)-expressing HEK293 cells. We also observed saturable uptake into human hepatocytes. Based on our estimation, the relative contribution of OATP1B1 to the uptake of valsartan in human hepatocytes depends on the batch, ranging from 20 to 70%. Regarding efflux transporters, the ratio of basal-to-apical transcellular transport of valsartan to that in the opposite direction in OATP1B1/MRP2 (multidrug resistance-associated protein 2) double transfected cells was the highest among the three kinds of double transfectants, OATP1B1/MRP2, OATP1B1/multi-drug resistance 1, and OATP1B1/breast cancer resistance protein-expressing MDCKII cells. We observed saturable ATP-dependent transport into membrane vesicles expressing human MRP2. We also found that the elimination of intravenously administered valsartan from plasma was markedly delayed, and the biliary excretion was severely impaired in EHBR compared with normal Sprague-Dawley rats. These results suggest that OATP1B1 and OATP1B3 as the uptake transporters and MRP2 as the efflux transporter are responsible for the efficient hepatobiliary transport of valsartan.