Although silybin serves as a well-known hepatoprotective agent with prominent anti-inflammatory, anti-oxidant and anti-fibrotic activities, its low bioavailability limits its application in the treatment of chronic liver diseases. However, novel formulation products with increased solubility were not sufficient to achieve pharmacologically meaningful concentrations of silybin in the clinical studies even used at high dosage. We hypothesized that inhibiting efflux transporter(s) and/or glucuronidation by piperine might enhance the bioavailability and efficacy of silybin. Pharmacokinetics of silybin given alone or in-combination with piperine was determined by a validated LC-MS method. A CCl4 induced rat model of liver injury was prepared and verified for comparing the effects of silybin and combination treatment. To investigate the underlying mechanism, the inhibition effects of piperine on transportation of silybin were performed in Caco-2 and transfected MDCKII cell lines as well as sandwich-cultured rat hepatocytes (SCH). Human liver microsomes incubation was used for exploring the modulation effects of piperine on the phase-2 metabolism of silybin. In the present study, we demonstrated for the first time that piperine as a bioenhancer increased the bioavailability of silybin (146%- 181%), contributing to a boosted therapeutic effect in CCl4-induced acute liver-injury rat model. The underlying mechanisms involved that piperine enhanced the absorption of silybin by inhibiting the efflux transporters including MRP2 and BCRP but not MDR1 in Caco-2 and transfected MDCKII cell lines. Moreover, piperine could inhibit the biliary excretion of silybin and conjugated metabolites in sandwich-cultured rat hepatocytes. Notably, we found that piperine did not affect the phase-2 metabolism of silybin. Efflux transporters play an important role in the pharmacokinetic behavior of flavolignans, and modulating these transporters by bioenhancer such as piperine could enhance the in vivo absorption of silybin, leading to more effective treatments. Copyright © 2018 Elsevier GmbH. All rights reserved.