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Molting is a key biological process of crustaceans, which is mainly regulated by 20-hydroxyecdyone (20E). The molting cycle could be divided into three main stages including pre-molt, post-molt and inter-molt stages. The mechanism of immune regulation during molting process still requires further exploration. Yorkie (Yki) is a pivotal transcription factor in the Hippo signaling pathway, and it plays an essential role in regulating cell growth and immune response. In the present study, a Yki gene was identified from Eriocheir sinensis (designed as Es Yki), and the regulatory role of Es Yki in controlling the expression of antimicrobial peptide genes throughout the molting process was investigated. The mRNA expression level of Es Yki was higher at the pre-molt stage compared to the post-molt stage and inter-molt stage. Following the injection of 20E, there was a notable and consistent rise in the Es Yki mRNA expression in haemocytes. The increase was observed from 3 h to 48 h with the maximum level at 12 h. And the phosphorylation of Yki in the haemocytes was also significantly up-regulated at 3 h post 20E injection. Moreover, the levels of Es Yki mRNA expression at three molting stages were significantly increased post Aeromonas hydrophila stimulation. The maximum level was detected at post-molt stage following A. hydrophila stimulation, while the lowest level was observed at inter-molt stage. The expression pattern of Es Crus was in contrast to Es Crus. After Es Yki mRNA transcripts were inhibited by Yki inhibitor (CA3), the mRNA expression levels of Es Crus1 and Es Crus2 following A. hydrophila stimulation were significantly elevated. Furthermore, the phosphorylation level of NF-κB was also increased following the inhibition of Yki. Collectively, our findings indicated that Es Yki could be induced by 20E and has a suppressive effect on the expression of Es Crus via inhibiting NF-κB during molting process. This research contributes to the understanding of the immunological regulation mechanism during molting process in crustaceans. • The mRNA expression level of Yki was the highset at the pre-molt stage. • 20-hydroxyecdysone could up-regulate the mRNA expression and phosphorylation levels of Yki. • Aeromonas hydrophila infection up-regulated the Yki mRNA expression during molting process of mitten crab. • Yki inhibition could promote Crustin mRNA expression. • The phosphorylation level of NF-κB was increased after inhibiting Yki. [ABSTRACT FROM AUTHOR]