No vaccine is yet commercially available against Mycobacterium marinum, the etiological agent of fish mycobacteriosis (also known as “fish tuberculosis”). The mycobacterial gene responsible for invasion and intracellular persistence, iipA, is known to moderate M. marinumpathology in Zebrafish Danio rerio. Two doses of heat‐killed, wild‐type, virulent M. marinumand two doses of a heat‐killed, avirulent M. marinum iipA::kanmutant strain were used in parallel to vaccinate European Seabass Dicentrarchus labrax. The fish were then challenged with live, virulent M. marinum, and the pathogenesis of the infection was monitored. High specific immunoglobulin M (IgM) response and an increase in cytokine tumor necrosis factor alpha (TNF‐α) messenger RNAexpression levels were observed in all vaccinated fish. At 1 month postchallenge, TNF‐α expression levels increased in spleen tissues of fish vaccinated with the virulent type and in those of unvaccinated fish, whereas in the head kidney, expression was up‐regulated only in unvaccinated fish. The expression then decreased, and at 2 months postchallenge, expression appeared similar in all vaccination types. The highest survival rate (75%) was recorded in the group of fish that were vaccinated with a high dose of avirulent iipA::kanmutant. The iipA::kanmutant induced a strong immune response accompanied by only modest tissue disruption. Coupled with an effective program of booster treatments, the iipA::kanmutant vaccine may be developed into a powerful preventive measure against fish mycobacteriosis.