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Subcellullar localizations and cross-immunities of GAPDHs from six common pathogenic bacteria in aquaculture were investigated.Subcellullar localizations of GAPDHs of Edwardsiella tarda EIB202, Edwardsiella ictaluri ATCC33202, Aeromonas hydrophila LSA34, Vibrio anguillarum MVM425, Vibrio alginolyticus EPGS020401 and Vibrio harveyi VIB647 were analysed with Western blotting, indirect immunofluorescence and flow cytometry examinations. Immunoprotections of different recombinant GAPDHs against these pathogens were investigated with zebrafish model. Western blotting of subcellular extractions showed that all GAPDHs were secreted into extracellular medium and periplasmic space. In addition, GAPDHs were demonstrated to distribute in the outer membranes except MVM425 and VIB647. And, GAPDHs were confirmed to be present on the surface of these bacteria with indirect immunofluorescence and flow cytometry examinations. The remarkable cross-protective immunities of these recombinant GAPDHs were induced in zebrafish, and the relative protective survivals were almost over 60%.Localizations of GAPDHs from these pathogenic bacteria were similar to many other causative agents. And, GAPDHs could be important protective antigens and give remarkable cross-immunity against different pathogens.Recombinant GAPDH could be designed as a broad spectrum vaccine candidate against multiple microbial infections in aquaculture.