Chitin synthase is an essential enzyme of the chitin synthesis pathway during molting. In this study, we identified and characterized a chitin synthase (EsCHS) gene in the Chinese mitten crab, Eriocheir sinensis. The spatio-temporal expression and functional role of EsCHS were investigated. The open reading frame of EsCHS was 4725 bp long and encoded 1574 amino acid residues that contained the typical domain structure of the glycosyltransferase family 2. Phylogenetic analysis revealed that EsCHS belongs to the group I chitin synthase family. The expression of EsCHS was found in regenerative limbs, the cuticle and the intestines. During the molting cycle, EsCHS began to increase in the pre-molt stage and reached a significant peak in the post-molt stage. The knockdown of EsCHS resulted in the significant downregulation of chitin biosynthesis pathway genes, including TRE, HK, G6PI, PAGM and UAP. Moreover, the long-term RNAi of EsCHS resulted in thinning procuticles, abnormal molting and high mortality, suggesting that EsCHS is indispensable for the formation of chitin in the cuticle during molting. In conclusion, EsCHS is involved in the chitin biosynthesis pathway and plays an important role in molting in E. sinensis. These findings highlight the potential of incorporating EsCHS into selective breeding programs to optimize molting regulation and improve growth performance in crustacean aquaculture.