In this study, by employing a unique approach of tandem affinity purification combined with MS analysis against a Tupaia hepatocyte proteome database established by deep sequencing, we revealed that the liver bile acid transporter, NTCP, specifically interacts with a key region in the pre-S1 domain of the HBV envelope L protein. By performing a series of virological analyses, we showed that silencing NTCP expression markedly inhibited viral infection of HBV and HDV in Tupaia as well as human hepatocytes. Exogenous expression of NTCP rendered nonsusceptible human hepatoma cells susceptible to the viral infections. The authentic viral infections in cells complemented with NTCP were shown by the kinetic analyses of several markers of viral infections, in particular the quantification of newly synthesized viral replicative intermediates. Moreover, the NTCP-rendered infections were blocked by known entry inhibitors. NTCP residues 157 to 165 were identified to be critical for pre-S1 binding and viral infections. These data clearly demonstrate that NTCP is a functional receptor for both HBV and HDV.