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The giant freshwater prawn, <i>Macrobrachium rosenbergii</i>, is an important and economical aquaculture species widely farmed in tropical and subtropical areas of the world. A new disease, “water bubble disease (WBD)”, has emerged and resulted in a large loss of <i>M. rosenbergii</i> cultured in China. A water bubble with a diameter of about 7 mm under the carapace represents the main clinical sign of diseased prawns. In the present study, <i>Citrobacter freundii</i> was isolated and identified from the water bubble. The optimum temperature, pH, and salinity of the <i>C. freundii</i> were 32 °C, 6, and 1%, respectively. A challenging experiment showed that <i>C. freundii</i> caused the same typical signs of WBD in prawns. Median lethal dose of the <i>C. freundii</i> to prawn was 10^4.94 CFU/g. According to the antibiogram tests of <i>C. freundii</i>, florfenicol and ofloxacin were selected to evaluate their therapeutic effects against <i>C. freundii</i> in prawn. After the challenge with <i>C. freundii</i>, 86.67% and 72.22% survival of protective effects against <i>C. freundii</i> were evaluated in the oral florfenicol pellets and oral ofloxacin pellets feding prawns, respectively, whereas the mortality of prawns without fed antibiotics was 93%. After antibiotic treatment and <i>C. freundii</i> infection, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), malondialdehyde (MDA), acid phosphatase (ACP), alkaline phosphatase (ALP), and lysozyme (LZM) in the hemolymph and hepatopancreas of the prawns and the immune-related gene expression levels of Cu/Zn-SOD, CAT, GPx, GST, LZM, ACP, anti-lipopolysaccharide factor, crustin, cyclophilin A, and C-type lectin in hepatopancreas were all significantly changed, indicating that innate immune responses were induced by <i>C. freundii</i>. These results can be beneficial for the prevention and control of <i>C. freundii</i> in prawns.