Fowl aviadenoviruses (FAdVs) are distributed worldwide in poultry farms. Some FAdVs are the causative agents of inclusion body hepatitis and hydropericardium syndrome that cause significant economic losses to the poultry industry. In contrast with human adenovirus, the study of the molecular biology of FAdV is still far behind. We previously showed that FAdV-9 open reading frame 1 (ORF1) is a dUTPase enzyme that contributes to the upregulation of type I interferons and is not required for virus replication in vitro. In the present study, we compared virus replication in vivo and the host immune response in chickens orally inoculated with a dUTPase knockout virus (ORF1stop), the rescued version of ORF1stop (resORF1), and wtFAdV-9. Our data showed that replication of ORF1stop was delayed on days 1 and 3 postinoculation compared with wtFAdV-9, as evidenced by significantly less virus shedding in feces and lower viral loads in tissues. Moreover, we found that there was a significant difference in the induction of cytokine gene mRNA expression in tissues and IgG antibody responses in ORF1stop versus wtFAdV-9-infected chickens, suggesting that ORF1 plays some roles in modulating the host immune response. Our study provides useful data on the mechanism of the host immune response against FAdV infection.