Mycobacteriosis, caused by numerous Mycobacterium spp., can be a devastating disease of both wild and cultured fishes. As no efficacious treatment exists, a vaccine against fish mycobacteriosis is essential for prevention and control of this disease. Thus, a DNA vaccine was constructed using the Mycobacterium marinum Ag85A gene that encodes one of the major secreted fibronectin-binding proteins of Mycobacterium spp., which was isolated and then subcloned into a commercially available eukaryotic expression vector. Juvenile hybrid striped bass (Morone saxatilis x M. chrysops), a species known to be particularly susceptible to this disease, were immunized by i.m. and i.p. injection with the resulting construct and as a result produced specific immune responses towards the Ag85A. Increasing concentrations of humoral antibodies to the Ag85A antigen were generated in all DNA vaccine groups, while macrophage phagocytosis and respiratory burst functions failed to exhibit upregulation after vaccination. In addition, fish receiving the DNA vaccine developed a protective response to a live M. marinum challenge 90 days post-inoculation, as demonstrated by increased survival of vaccinated fish over control fish and by reduced splenic bacterial counts in vaccinated fish. Furthermore, humoral immune responses and protective effects were significantly increased at higher vaccine doses using the i.m. injection route.