Background: Hepatitis B virus (HBV) infection can lead to end-stage cirrhosis and hepatocellular carcinoma (HCC). Although approved antiviral drugs strongly inhibit viral replication, long-term treatment leads to drug-resistant variants and adverse side effects. Hence, the treatments of patients with HBV and virus carriers are likely to remain a major health problem for decades to come. The aim of this study was to investigate the effects of the distal upstream binding factor-far upstream element binding protein 1 (FUBP1) on the activities of the core promoter and enhancer II (EnhII)/basic core promoter (BCP) in Huh7 cells infected with HBV. In addition, we investigated the molecular mechanism of FUBP1 regulation of HBV transcription to find new targets for anti-HBV therapy. Methods: The FUBP1 overexpression plasmid with HA label (pcDNA3.1-HA-FUBP1) was transfected and overexpressed in Huh7 cells. Western blot analysis was used to verify successful construction of the FUBP1 overexpression plasmid, as well as the overexpression of FUBP1. Co-transfection of pcDNA3.1-HA-FUBP1 and Plasmids containing the 1.24-fold HBV genomes (pUC19-HBV-Ce), together with real-time fluorescence quantitative PCR (RT-qPCR), was used to investigate the effects of FUBP1 overexpression on HBV pre-genomic RNA (pgRNA), spliced RNA (spRNA), and splicing efficiency. The results of RT-qPCR were further verified by Northern blot analysis. The poly(U)-binding splicing factor 60 kDa (PUF60) and FUBP1 are known to form a complex that exerts regulatory functions. Hence, the effects of PUF60, FUBP1, and their co-expression on HBV pgRNA level, pg promoter activity, and EnhII/BCP core promoter activity were investigated by RT-qPCR and sea kidney luciferase reporting assays. Alpha screen and RNA degradation experiments were also used to examine whether FUBP1 regulation of RNA transcription was related to post-transcriptional RNA extranuclear output and degradation. A series of HBV promoters and mutants were constructed, and the effects of FUBP1 overexpression on the activity of these HBV promoters and their mutants were evaluated. Gel migration assay was used to further investigate the targeting of the HBV EnhII/BCP region by FUBP1. Results: Overexpression of FUBP1 significantly decreased the expression levels of pgRNA (p < 0.01) and spRNA (p < 0.001), and decreased the splicing efficiency (p < 0.001). These results were further verified by Northern blot. FUBP1 inhibited HBV transcription by down-regulating the activity of HBV EnhII/BCP (p < 0.001). Previous studies have reported that PUF60 often forms a complex with FUBP1. The present study found that FUBP1 independently targets the HBV EnhII/BCP region to inhibit the expression of HBV pre-genomic RNA (p < 0.001). Further studies showed that inhibition of HBV pgRNA by FUBP1 was not achieved by post-transcriptional regulation of extranuclear export and degradation of HBV RNA. FUBP1 regulates promoter activity by targeting promoters with the common sequence of del-1 (deletion of nt 1627—1652) and del-2 (deletion of nt 1640—1674) region nt1640–1652 (specific base sequence: GCCCAAGGTCTTG), thereby inhibiting HBV replication. Conclusions: FUBP1 binds directly to the HBV nt1640-1652 region to down-regulate promoter activity and inhibit HBV transcription. FUBP1 plays a negative role in HBV transcription and is a potential target for anti-HBV therapy.