Entecavir (ETV) is a first-line antiviral agent for the treatment of chronic hepatitis B virus infection. Renal excretion is the major elimination path of ETV, in which tubular secretion plays the key role. However, the secretion mechanism has not been clarified. We speculated that renal transporters mediated the secretion of ETV. Therefore, the aim of our study was to elucidate which transporters contribute to the renal disposition of ETV. Our results revealed that ETV (50 M) remarkably reduced the accumulation of probe substrates in MDCK cells stably expressing human multidrug and toxin efflux extrusion proteins (hMATE1/2-K), organic cation transporter 2 (hOCT2), and carnitine/organic cation transporters (hOCTNs) and increased the substrate accumulation in cells transfected with multidrug resistance-associated protein 2 (hMRP2) or multidrug resistance protein 1 (hMDR1). Moreover, ETV was proved to be a substrate of the above-described transporters. In transwell studies, the transport of ETV in MDCK-hOCT2-hMATE1 showed a distinct directionality from BL (hOCT2) to AP (hMATE1), and the cellular accumulation of ETV in cells expressing hMATE1 was dramatically lower than that of the mock-treated cells. The accumulation of ETV in mouse primary renal tubular cells was obviously affected by inhibitors of organic anion transporter 1/3 (Oat1/3), Oct2, Octn1/2, and Mrp2. Therefore, the renal uptake of ETV is likely mediated by OAT1/3 and OCT2 while the efflux is mediated by MATEs, MDR1, and MRP2, and OCTN1/2 may participate in both renal secretion and reabsorption. C hronic hepatitis B virus (HBV) infection, with a high rate of morbidity, is one of the most important health problems worldwide. It is a major risk factor for cirrhosis and liver cancer (1). Entecavir (ETV) is a novel and highly selective deoxyguanosine analog with a high antiviral efficacy and a high genetic barrier to viral resistance (2). Since ETV was approved in 2005 by the U.S. FDA, it has been unanimously recommended as the fist-line antiviral agent for treatment of chronic HBV infection by global hepatology scientific societies, such as the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver (3, 4).It was reported that about 73% of orally dosed ETV was eliminated in urine in an unchanged form. The renal clearance of ETV is independent of the dose and ranged from 360 to 471 ml/min in healthy volunteers, which is much greater than the glomerular filtration rate (GFR; 120 to 130 ml/min for normal humans) (5, 6), suggesting that tubular secretion accounts for the major part of the urinary excretion of ETV. Therefore, it is speculated that transporters expressed on the proximal tubular cells are involved in the process of tubular secretion of ETV. Furthermore, ETV is a hydrophilic weak base with a pK a value of 10.5 (Ͼ99% of ETV is positively charged at pH 7.4) and a logD value of Ϫ1.1 (pH 4 to 10), which implies that ETV is unlikely to penetrate the cell membrane by passive diffusion. Thus, transpor...