Abstract: The purpose of this study was to explore the innate immune response and immune regulation mechanism of SPF chickens infected with Fowl adenovirus serotype 1（FAdV-1）. The results showed that there was no obvious morbidity and death in the observation group, infected group and control group of SPF chickens infected with FAdV-1, and slight pathological changes were found in the muscular stomach and glandular stomach. In the early stage of infected, the expressions of Toll-like receptors 2（TLR2）, 5, 7, 21, avian β-defensins 5（AvBD5）, 8, Inter leukin-8（IL-8）, 18, Myeloid differentiation factor 88（My D88）, C-Rel and p65 genes in small intestinal tissue were significantly increased. The expression trends of TLR2, TLR5, AvBD8, IL-8, MyD88, C-Rel and p65 were consistent in 36 hpi. In the late stage of infected, the expressions of TLR15, AvBD10, 12 and IL-8 genes were significantly up-regulated in some tissues. The results showed that the innate immune response of SPF chickens infected with FAdV-1 was produced through TLR2-MyD88 and TLR5-MyD88 pathway in the small intestine and regulated AvBDs and cytokines to eliminate the virus. This experiment provided an important theoretical basis for improving the innatel immune function of poultry, and for preventing and controlling FAdV-1 and similar pathogens through exogenous conditions in the future.