HBoV1, an autonomously replicating human parvovirus, causes acute respiratory tract infections in young children. HBoV1 infects and productively replicates in non-dividing (well-differentiated) cells of HAE-ALI. HBoV1 replication induces a DNA damage response and activates DNA repair, which drive viral genome amplification. In this report, we identified that NP1, a small nonstructural protein encoded in the center of the viral genome, directly interacts with Ku70 and RPA70 at a high affinity. The key interaction domains of Ku70 and RPA70 were characterized and confirmed by the inhibition of HBoV1 viral DNA replication both in a newly established in vitro viral DNA replication assay and in HBoV1-infected HAE-ALI. Thus, our study demonstrated that the interactions of NP1 with Ku70 and RPA70 enhance HBoV1 DNA replication, highlighting the essential role of the small nonstructural protein in parvoviral DNA replication and the importance of the DNA repair factors in viral DNA replication.