We previously reported the development of an inactivated oil-adjuvanted avian influenza vaccine using an apathogenic H5N1 strain of the same lineage as the Eurasian lineage viruses currently epidemic in Asia. In this study, we confirmed the safety and evaluated the efficacy of this vaccine in layer chicken farms by field trials. No problematic adverse reactions occurred in the safety test. In addition, no adverse effects were observed in the field trial, and the antibody titer exceeded a protective level (hemagglutination inhibition (HI) antibody titer of 16) at 3 weeks after a single injection. Based on the above findings, this vaccine was confirmed to be safe and induced a protective level of antibody titer with a single injection in the chickens at the farms.