Chitinase, belonging to family 18 glycosyl hydrolase, is a multi-gene family and it has many functions. Generation of loss-of-function mutant targeting an interesting gene is a common way to clarify its function based on reverse genetics. In this study, we first reported the immune defense of a chitinase gene (EcChi4) in Exopalaemon carinicauda using its EcChi4-deletion mutant. EcChi4 was predominantly expressed in hepatopancreas and was upregulated after challenge with Vibrio parahaemolyticus or Aeromonas hydrophila. After knockout EcChi4 gene using CRISPR/Cas9 tool, the prawns in EcChi4-deletion group had significant higher mortality than those in wild-type group when the prawns were challenged with V. parahaemolyticus or A. hydrophila. In conclusion, we first demonstrate the function of a chitinase gene in immune defense of E. carinicauda by performing directed, heritable gene mutagenesis. In the future, CRISPR/Cas9 should be widely applicable as a feasible means for gene editing in E. carinicauda for the study of important biological questions that cannot be easily addressed in other decapods.