HepG2 and Huh7 cell lines are frequently used as models to study viral hepatitis and hepatocellular carcinoma. However, they exhibit significantly different capacities in their ability to support hepatitis B virus (HBV) replication. To investigate the basis for this, transcription factor–binding motifs at the HBV core promoter (HBVCP) were tested in luciferase reporter constructs to identify the possible role of host factors. Among the transcription factors screened: PARP1, SP1, HNF4α, HNF3, hB1F and HNF1, deletion of the PARP1 binding motif abrogated transcriptional activity at the HBVCP in HepG2 but not Huh7 cells. Sequencing of the PARP1 gene revealed that HepG2 cells carried an Ala762 allele which has low ADP-ribosylation activity, which was shown to have increased PARP1 binding affinity to its cognate motif thus resulting in higher transcriptional activity. PARP1 inhibitors that are being developed as broad cancer therapeutics also target PARP1 ADP-ribosylation enzymatic function. Four PARP1 inhibitors: PJ-34, ABT888, AZD2281 and AG014699 were tested for their effect on HBV replication. All four small molecules effectively enhanced HBV replication in vitro, confirming the role of PARP1 in HBV replication and that alteration of ADP-ribosylation by mutation or drugs can affect HBV replication. Our data demonstrate that natural polymorphisms in the host affecting proteins such as PARP1 can have a significant effect on HBV replication. Hence, patients who are infected with HBV and are on clinical trials involving PARP1 inhibitors may be at risk from unintended side-effects such as exacerbation of HBV replication.