Transcription, replication, and segregation of human papillomaviruses (HPVs) are regulated by various host factors, but our understanding of host proteins that bind to the HPV genome is limited. Here we report the results of a search of cellular proteins that can associate with specific genomic regions of HPV type 16 (HPV16). We found that human nucleolin, an abundant nucleolar protein, was preferentially captured in vitro by an HPV16 genomic fragment from nucleotide positions (nt) 531-780. Electrophoretic mobility shift assays with a bacterially expressed nucleolin revealed that nucleolin bound to an HPV16 genomic region between nt 604 and 614 in a sequence-dependent manner. Chromatin immunoprecipitation analysis showed that both exogenous and endogenous nucleolin bound to a plasmid containing the HPV16 genomic region in HeLa cells. Furthermore, nucleolin associated with the HPV16 genome stably maintained in HPV16-infected W12 cells, suggesting that the nucleolin binding may be involved in the dynamics of the HPV genome in cells.