When human adenovirus infects human cells there is disruption of rRNA biogenesis. This report examines the effect of adenovirus infection on the nucleolar protein, upstream binding factor (UBF) which plays a major role in regulating rRNA synthesis. We determined that early after infection, UBF associates with the replication of viral DNA, preferentially associating with the ends of the linear viral genome, and that addition of anti-UBF serum to in vitro replication assays markedly reduced viral DNA replication. Regions of UBF important to these observations are also established. Interestingly, sequestering the majority of UBF from the nucleolus did not lead to the ablation of rRNA synthesis or the sequestration of RNA pol I. In infected cells the bulk of RNA synthesis was RNA pol I associated and distinct from the location of most of the detectable UBF. We propose that UBF plays a role in viral DNA replication, further strengthening the role of nucleolar antigens in the adenovirus life cycle.