Small DNA viruses such as hepatitis B virus (HBV) and adeno-associated virus (AAV) rely extensively on host cellular proteins and pathways to establish productive infections. The formation and maintenance of episomal transcriptional templates are key determinants of HBV persistence and the success of AAV-based gene therapy approaches. However, the production and expression of these episomes are rate-limiting steps in the replication cycles and are primarily regulated by host factors, many of which remain poorly characterized. Previously, our laboratory identified several cellular factors that negatively regulate HBV infection through a loss-of-function genetic screen. In the present study, we characterize the role of the Ku70/Ku80 heterodimer, two of the hits from that screen. Silencing of Ku70 or Ku80 led to increased viral gene expression at both mRNA and protein levels upon HBV infection, confirming their function as negative regulators. Similar effects were observed in AAV vector-transduced cells, indicating a broad-spectrum restrictive effect. Co-expression of wild-type Ku70 and Ku80 rescued the silencing phenotype, while mutants mislocalized to the cytoplasm or defective in DNA binding did not, suggesting that restriction occurs in the nucleus and is independent of Ku's DNA binding capacity. Mechanistic analyses by means o ChIP-qPCR revealed that Ku70 silencing does not alter cellular RNA polymerase II recruitment to the AAV genome, excluding transcriptional regulation as the targeted step. In contrast, Southern blot and qPCR assays demonstrated increased accumulation of viral episomal DNA in Ku70-silenced cells, suggesting that Ku70 negatively regulates viral episomal DNA content. In summary, our findings indicate that the Ku70/Ku80 heterodimer restricts the accumulation of viral episomal transcriptional templates, likely by influencing their formation and/or stability.