Hepatitis B virus (HBV) is an important human pathogen, which targets the liver extremely efficient, gaining access to hepatocytes by a so far unknown receptor and replicating in a hepatocyte-specific fashion. Cell differentiation seems to determine HBV replication. We here show that the level of hepatocyte differentiation, as indicated by hepatocyte polarization and metabolic activity, is closely correlated to the transcription of the HBV RNA pregenome. Pregenome transcription determined the level of HBV replication in various cell lines of hepatocellular origin and in primary human hepatocytes. A variety of hepatocyte-enriched nuclear factors have been described to regulate transcription of the pregenome, but it remained unknown which factors link HBV replication to hepatocyte differentiation. We determined that high expression levels of HNF4α but not its potential cofactors or other hepatocyte-enriched transcription factors were essential for efficient HBV replication, and link it to hepatocyte differentiation. HNF1α contributed to the control of HBV replication because it regulated the expression of HNF4α. Thus, a concerted action of HNF4α and HNF1α, which also determines morphological and functional differentiation of hepatocytes, links HBV replication to hepatocyte differentiation.