Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. HBoV1 productively infects polarized primary human airway epithelia. However, no cell lines permissive to HBoV1 infection have yet been established. More importantly, the sequences at both ends of the HBoV1 genome have remained unknown. We have resolved both of these issues in this study. We have sequenced a full-length HBoV1 genome and cloned it into a plasmid. We further demonstrated that this HBoV1 plasmid replicated and produced viruses in human embryonic kidney 293 cells. Infection of these HBoV1 progeny virions produced obvious cytopathogenic effects in polarized human airway epithelia, which were represented by disruption of the epithelial barrier. Moreover, we identified an airway epithelial cell line supporting HBoV1 infection, when it was polarized. This is the first study to obtain the full-length HBoV1 genome, to demonstrate pathogenesis of HBoV1 infection in human airway epithelia, and to identify the first cell line to support productive HBoV1 infection.