A fully effective treatment of chronic human hepatitis B virus (HBV) infection is still missing and HBV remains the first etiological agent of liver cancer. Although the viral regulatory X protein is essential for infection, its mode of action remains obscure, due the lack of an in vitro infection system. In the accompanying study, we showed the functional importance of interaction between X and the host protein UVDDB-p127, in the transactivation and apoptotic properties of the viral protein. Here, we addressed the biological role of X-UVDDB interaction in the infectious process using a genetic approach in the woodchuck virus closely related to HBV. We show that (i) mutations in X, which markedly affect UVDDB-binding, also abolished productive infection in woodchucks,(ii) in the few cases where mutant viruses led to infection, compensatory mutations had occurred in the X gene of the viral progeny, which restored correct UVDDB-binding. We conclude that efficient viral replication in vivo requires proper X-UVDDB interaction. The interaction may thus provide a novel therapeutic target for the treatment of hepatitis B.