Hepatitis-hydropericardium syndrome (HHS) caused by hypervirulent fowl adenovirus 4 (FAdV-4) have been causing great economic losses to Chinese poultry industry since 2015. Elucidation of the pathogenesis of FAdV-4 will lay solid foundation for developing attenuated FAdV-4 vaccine and vaccine vector. Our previous study has demonstrated that the increased virulence of hypervirulent FAdV-4 was associated with fiber-2 and hexon genes. However, the roles of fiber-1 and penton in virulence of FAdV-4 have never been elucidated. To further investigate the roles of the major structural proteins fiber-1 and penton in the virulence of hypervirulent FAdV-4, the fiber-1- and penton-replaced mutant viruses were constructed based on the FAdV-4 infectious clones of hypervirulent strain HNJZ using Redαβ recombineering techniques. The pathogenicity of the rescued viruses was evaluated in 3-week-old SPF chickens. Chickens infected with the rescued recombinant viruses carrying the fiber-1 or penton base gene from a nonpathogenic strain ON1 developed similar clinical signs to the natural hypervirulent FAdV-4 infection, including HHS-indicative gross lesions and histopathological changes in sick/dead chickens. Our results suggested that the increased virulence of hypervirulent FAdV-4 was independent of fiber-1 and penton. The detailed pathogenesis of FAdV-4 and the roles of fiber-1 and penton in the viral replication and infection process need to be further explored.Copyright © 2020. Published by Elsevier Ltd.