Fowl adenovirus serotype-4 (FAdV-4) infection, also known as hydropericardium-hepatitis syndrome (HHS), is one of the most prevalent diseases in the Korean poultry industry. Therefore, an effective vaccination against FAdV-4 should be developed for prevention of HHS infection. However, a standard animal model with significant pathologic characteristics has not been established for evaluation of HHS vaccine efficacy. In this study, we comparatively evaluated FAdV-4 pathogenicity from specific- pathogen-free (SPF) chickens using FAdV-4 strain (ADL091024) isolated in laying chickens in Korea according to chicken age, passage number and virus titers. In brief, FAdV-4 passaged four times in yolk sac of SPF eggs with the highest viral titers could induce the most severe clinical signs in 1-day-old chickens, similar to natural HHS outbreaks. Furthermore, the efficacy of a newly developed attenuated live FAdV-4 vaccine candidate was successfully determined using the established 1-day-old SPF chickens. There was no significant pathogenicity based on the criteria such as mortality, body weight, gross lesions, histological lesions and virus detection rates in the vaccination and non-challenge control groups. However, the challenge group without vaccination showed significant pathogenicity including hepatic necrosis, histological lesion scores in the liver and heart, virus replication and decreased body weight gain. In conclusion, the chicken challenge model established in this report was proposed for consistent and reliable evaluation of FAdV- 4 vaccine efficacy.