BACKGROUND AND AIMS: Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and hepatocellular carcinoma. Hypoxia inducible factors (HIFs) are a hallmark of inflammation and are key regulators of hepatic immunity and metabolism and yet their role in HBV replication is poorly defined. HBV replicates in hepatocytes within the liver, a naturally hypoxic organ, however most studies of viral replication are performed under conditions of atmospheric oxygen, where HIFs are inactive. We therefore investigated the role of HIFs in regulating HBV replication. METHODS: Using cell culture, animal models, human tissue and pharmacological agents inhibiting the HIF-prolyl hydroxylases we investigated the impact of hypoxia on the HBV life cycle. RESULTS: Culturing liver cell-based model systems under low oxygen uncovered a new role for HIFs in binding HBV DNA and activating the basal core promoter, leading to increased pre-genomic RNA and de novo HBV particle secretion. The presence of hypoxia responsive elements among all primate members of the hepadnaviridae highlight an evolutionary conserved role for HIFs in regulating this virus family. CONCLUSIONS: Identifying a role for this conserved oxygen sensor in regulating HBV transcription suggests that this virus has evolved to exploit the HIF-signalling pathway to persist in the low oxygen environment of the liver. Our studies show the importance of considering oxygen availability when studying HBV-host interactions and provide innovative routes to better understanding and therapeutic targeting of chronic HBV infection.