In this study, we investigated the regulatory roles of the juvenile hormone epoxide hydrolase (JHEH) gene in the reproductive process of female Macrobrachium nipponense. Its total cDNA length was 1848 bp, encoding for 460 amino acids. It contained conserved domains typical of epoxide hydrolases, such as the Abhydrolase family domain, the EHN epoxide hydrolase superfamily domain, and the “WWG” and “HGWP” motifs. The qPCR results showed that the expression of Mn-JHEH was the highest in hepatopancreas. Mn-JHEH was expressed at all stages of the embryonic and larval stages. The expression of Mn-JHEH at different developmental periods of the ovary was positively correlated with ovarian maturation. In situ hybridization showed that it was mainly located in the cytoplasmic membrane and nucleus of oocytes. The RNA interference technique was used to study the role of Mn-JHEH in the process of ovarian maturation. The knockdown of Mn-JHEH with dsRNA in the experimental group resulted in a significant decrease in the percentage of ovaries exceeding stage O-III and the gonadal index compared with the control group. On day 14 (the second molt), the molt frequency was significantly higher in the control group than in the experimental group. The results showed that Mn-JHEH played an important role in ovarian maturation and molting.