FAdV-9Δ4, lacking six open reading frames (ORFs) 0, 1, 1A, 1B, 1C and 2 at the left end of FAdV-9 genome, shows potential as a vaccine vector. In vivo studies demonstrated that FAdV-9Δ4 does not replicate as efficiently as wild type (wt) virus in intramuscularly inoculated chickens, suggesting the important roles of the left-end ORFs. The fecal-oral route is the natural route of FAdV infection, and the oral administration confers several advantages compared to administration through other routes when developing a vaccine. Therefore, the objectives of this study were to investigate the effects of oral inoculation with both FAdV-9Δ4 and wtFAdV-9 in chickens and to explore the functions of one of the left-end ORFs, ORF1. Chickens were orally inoculated with FAdV-9Δ4 or wtFAdV-9. Compared to wtFAdV-9 group, reduced virus shedding in feces, lower viral loads in tissues, and lower antibody response were found in FAdV-9Δ4 group. There were also significant differences between FAdV-9Δ4 and wtFAdV-9 in terms of the induction of cytokine mRNA expression, including interferon α (IFN-α), IFN-γ, and interleukin-12 (IL-12). This indicated the important roles of the six ORFs in modulation of the host immune response. ORF1, a homolog of deoxyuridine 5′-triphosphate pyrophosphatase (dUTPase), was hypothesized to be important in virus replication and regulation of host immune response. In this project, FAdV-9 ORF1 was verified as a functional dUTPase and its molecular features were characterized, including transcription and protein expression patterns and cellular localization. A dUTPase knockout virus (ORF1stop) and its rescued revertant (resORF1) were generated. Functional studies showed that FAdV-9 dUTPase was not required for virus replication in vitro, but played a role in virus replication in vivo. FAdV-9 dUTPase also contributed to the regulation of the expression of IFN-α, IFN-β, and IFN-γ both in vitro and in vivo, and the host antibody response as well. This is the first study to functionally identify an early gene of FAdV-9. The data presented are helpful for better understanding of the molecular biology of FAdVs and for exploring the mechanism of the host immune response against FAdV infections.