In the present study, the pathogenicity of fowl adenovirus (FAdV) serotype 4 strain HB1501 was systematically determined by using different doses and routes of inoculation in chickens and assessed the immune efficacy induced by fowl adenovirus serotype 4 (FAdV-4) strain SB15 in a vaccination trial. The results revealed that strain HB1501 was highly virulent in chickens, inflicting 30 to 100% mortality when administered by intramuscular and intravenous routes, and its pathogenicity was influenced markedly by the inoculation route. The inactivated oil-emulsion FAdV-4 vaccine derived from strain SB15 completely protected specific pathogen-free chickens against morbidity and mortality following infection with the virulent FAdV-4 strain HB1501. Importantly, no obvious clinical signs or gross lesions were observed and high antibody levels against FAdV were measured in the SB15-vaccinated chickens, indicating the potential of this strain to become an alternative vaccine candidate. This study provides detailed information on the pathogenicity of FAdV-4, which should be of great value to the control of hepatitis-hydropericardium syndrome.