Adenovirus (Ad) cell attachment is initiated by the attachment of the fiber protein to a primary receptor (usually CAR or CD46). This event is followed by the engagement of the penton base protein with a secondary receptor (integrin) via its loop region, which contains an Arg-Gly-Asp (RGD) motif, to trigger virus internalization. To understand the well-orchestrated adenovirus cell attachment process that involves the fiber and the penton base, we reconstructed the structure of an Ad5F35 capsid, comprising an adenovirus type 5 (Ad5) capsid pseudotyped with an Ad35 fiber, at a resolution of approximately 4.2 Å. The fiber-penton base interaction in the cryo-electron microscopic (cryo-EM) structure of Ad5F35 is similar to that in the cryo-EM structure of Ad5, indicating that the fiber-penton base interaction of adenovirus is conserved. Our structure also confirms that the C-terminal segment of the fiber tail domain constitutes the bottom trunk of the fiber shaft. Based on the conserved fiber-penton base interaction, we have proposed a model for the interaction of Ad5F35 with its primary and secondary receptors. This model could provide insight for designing adenovirus gene delivery vectors.