Abstract: Nanocerium dioxide (nCeO₂) is an important new type of rare earth metal oxide with high oxidation resistance. In order to develop new antimicrobial agents witch are efficient, environmentally friendly and non-resistant, we obtained nCeO₂ from cerium nitrate hexahydrate by high temperature calcination. We sudied the in vitro antibacterial activity of nCeO₂ on two kinds of aquatic pathogens (Vibrio vulnificus and V. parahaemolyticus), and verified the antibacterial effect of nCeO₂ by in vivo experiment of Macrobrachium rosenbergii. The results show that: 1) The obtained nCeO₂ was relatively pure. The particle diameter was 25.03 nm, the Zeta potential (mV) was −5.65, and the dispersion in the solution was relatively uniform. 2) The minimum inhibitory concentrations (MIC) of nCeO₂ against V. vulnificus and V. parahaemolyticus were 128 and 256 μg·mL⁻¹, respectively, and the minimum bactericidal concentrations (MBC) all were 512 μg·mL⁻¹. The diameter of the inhibition ring was 21.50 mm for V. vulnificus and 17.42 mm for V. parahaemolyticus. 3) The three concentrations of nCeO₂ (1/2 MIC, 1 MIC and 2 MIC) all significantly improved the extracellular AKP activity, relative conductivity of bacterial solution, nucleic acid content and soluble protein content in the extracellular (P<0.05). 4) The nCeO₂ supplementation in the feed significantly reduced the mortality of M. rosenbrei in V. vulnificus and V. parahaemolyticus infection models (P<0.05). The results indicate that nCeO₂ has a good inhibitory effect on aquatic pathogens V. vulnificus and V. parahaemolyticus, and the antibacterial action may be related to its destruction of cell wall membrane integrity and cell membrane permeability.