Adenoviruses (AdVs) are large DNA viruses that can cause infections in almost all vertebrates. Mastadenoviruses cause infections in human and non-human primates where the members of the genus Atadenovirus, Siadenovirus, and Aviadenovirus predominantly infect avian hosts. AdVs replicate within the nucleus, but the mechanisms regulating viral DNA transport to the host nucleus during infection remain largely unsolved. The adenovirus core protein VII (pVII) binds viral DNA and is believed to play a major role in localizing viral DNA to the nucleus, but the mechanism is not understood. The study, therefore, aimed to reveal the structural interactions between the putative nuclear localization sequence (NLS) of pVII in Psittacine siadenovirus F (PsSiAdV-F) that infects one of the most critically endangered Australian parrots, orange-bellied parrot (Neophema chrysogaster) and the nuclear transport protein, importin alpha (IMPα). The putative NLS region of pVII of PsSiAdV-F was identified and complexed with IMPα, and the structure was determined using X-ray crystallography to 2.2 Å resolution. Structural characterization of the interface reveals that pVII binds to IMPα with a classical monopartite NLS, with a well-characterized lysine at the P2 site. Thus, identification of the critical residues within pVII for mediating IMPα binding supports further investigation into molecular targets in adenovirus systems to prevent or combat infections and developing adenovirus-based vectors for gene delivery.