Vibrio harveyi is one of the most common threats to farmed grouper, so considerable efforts are in practice to control the pathogen. This study presents a highly effective vaccine against V. harveyi in the orange-spotted grouper with the help of a single intraperitoneal immunization. The vaccine candidate was in form of whole, formalin-inactivated V. harveyi cells combined with a metabolizable ISA763 AVG adjuvant. Our results indicated that the vaccine triggered a remarkably higher expression level of interleukin (IL)-1β, IL-6, IL-8, and IL-10 in the groupers' kidneys and spleens, as recorded after 1 and 3 days of immunization. Antibody titers were significantly elevated in the vaccinated fish. A pivotal observation was that the vaccine highly protected the grouper from a homologous V. harveyi strain challenge with relative percentage survival values of 100% and 91.7% at 6 and 12 weeks post-immunization, respectively. Vaccinated fish also demonstrated strong cross-protection against a heterologous bacterial isolate challenge. Therefore, the inactivated V. harveyi vaccine is a promising candidate that could stimulate good immune responses and confer remarkable protection in farmed groupers.