Methyl farnesoate (MF), the crustacean juvenile hormone (JH), plays critical roles in various physiological processes in crustaceans. The titer of MF is precisely regulated by specific carboxylesterase. Here, we report for the first time that the cloning and expression analysis of a JH esterase-like carboxylesterase from the prawn Macrobrachium rosenbergii (named as MrCXE). MrCXE contained a 1935-bp open reading frame (ORF) conceptually translated into a 644-amino acids protein. MrCXE protein shared the highest identity (36%) with JH esterase-like carboxylesterase from the swimming crab, Portunus trituberculatus and exhibited the typical motifs of JH esterase-like carboxylesterases. MrCXE was most abundantly expressed in hepatopancreas, the major tissue for MF metabolism. MrCXE was expressed at a low level in gut and was not detected in other tissues. Additionally, MrCXE expression was upregulated in hepatopancreas by eyestalk ablation to increase MF level. Furthermore, the mRNA level of MrCXE was significantly increased in the hepatopancreas when challenged by the bacterial pathogens Aeromonas hydrophila and Vibrio parahaemolyticus. To our knowledge, this is the first report that the JH esterase-like carboxylesterase is involved in the innate immune response of the crustaceans.