Chitin deacetylases are essential enzymes in the chitin-modifying process and play vital roles in arthropod molting. In this study, we identified and characterized a chitin deacetylase-like (EsCDA-l) gene in the Chinese mitten crab, Eriocheir sinensis. The open reading frame of EsCDA-l was 2555 bp and encoded 554 amino acid residues that contained typical domain structure of carbohydrate esterase family 4. Phylogenetic analysis reveal that EsCDA-l belongs to the group I chitin deacetylase family. Quantitative real-time PCR analyses showed that EsCDA-l was highly expressed in exoskeletal tissues and megalopa stages. During the molting cycle, EsCDA-l was up-regulated periodically in the post-molt stage. Knockdown of EsCDA-l resulted in the abnormal ultrastructure of cuticle, prevented molting to high mortality suggesting EsCDA-l is indispensable for molting. The characterization and function analysis of the EsCDA-l should provide useful reference for further research on the utility of key genes involved in the chitin metabolic pathway in the molting process of the Chinese mitten crab as well as other crustaceans.