Background & Aims: Our understanding of the interactions between HBV and its host cells is still quite limited. Spliceosome associated factor 1 (SART1) has recently been found to restrict HCV. Thus, we aimed to dissect its role in HBV infection. Methods: SART1 was knocked down by RNA interference and over-expressed by lentiviral or adeno-associated virus (AAV) vectors in HBV-infected cell cultures and in vivo in HBV-infected mice. Luciferase reporter assays were used to determine viral or host factor promoter activities, and chromatin immunoprecipitation (ChIP) was used to investigate protein-DNA interactions. Results: In HBV-infected cell cultures, downregulation of SART1 did not affect covalently closed circular HBV DNA but resulted in markedly enhanced HBV RNA, antigen expression and progeny virus production. On the other hand, HBV transcription and replication were significantly inhibited by overexpression of SART1. Similar results were observed in AAV-HBV-infected mice persistently replicating HBV. Inhibition of Janus kinases had no effect on SART1-mediated inhibition of HBV replication. HBV promoter assays revealed that SART1 reduced HBV core promoter activity. By screening known HBV transcription factors, we found that SART1 specifically suppressed the expression of hepatocyte nuclear factor 4 alpha (HNF4 alpha). Luciferase reporter and ChIP assays demonstrated a direct downregulation of HNF4 alpha expression by association of SART1 with the HNF4 alpha proximal P1 promoter element. Conclusions: We identify SART1 as a novel host factor suppressing HBV cccDNA transcription. Besides its effect on interferon-stimulated genes, SART1 exerts an anti-HBV activity by suppressing HNF4 alpha expression, which is essential for transcription of HBV cccDNA. Lay summary: Hepatitis B virus (HBV) infects hepatocytes and persists in the form of covalently closed circular DNA (cccDNA), which remains a major obstacle to successful antiviral treatment. In this study, using various HBV models, we demonstrate that the protein SART1 restricts HBV cccDNA transcription by suppressing a key transcription factor, HNF4 alpha. (C) 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.