In order to explore the regulatory effect of microRNA (miRNA) on Macrobrachium nipponense chitinase 3A(MnCHT3A) gene, bioinformatics approach was firstly used to predict and screen the miRNA--miR-305-5p bound specifically to MnCHT3A. Using qRT-PCR, biochemical and histological methods, the regulation of miR-305-5p on target gene MnCHT3A was studied in vivo. The results showed that the expression change of miR-305-5p was negatively correlated with MnCHT3A during the molting cycle of M. nipponense. The level of miR-305-5p peaked at stage C and was the lowest at stage A, while the expression trend of MnCHT3A mRNA was opposite. After injection of miR-305-5p mimics or miR-305-5p inhibitor, the transcription level of MnCHT3A was decreased by 60% or increased by 166%, and the activity of MnCHT enzyme, meanwhile, was decreased by 39.53% or increased by 133%, respectively compared with the control group. Histological results showed that a three-layer cuticle structure of carapace in stage C was observed by means of H-E staining, namely, epicuticle, exocuticle and endocuticle from outside to inside. The images of chitin fluorescence staining showed the presence of chitin in the exocuticle and endocuticle. Results from scanning electron microscopy clearly showed the lamellar structure of the exocuticle and endocuticle. Compared with the control group, a thickening trend in the endocuticle with the lamellae as well as the blue fluorescence chitin stripe was observed in miR-305-5p mimics group. But in miR-305-5p inhibitor group, the culticular structure was disordered. Correspondingly, the blue fluorescence chitin stripe was not uniform and weakened in some areas. The results obtained above indicate that the target gene of miR-305-5p is MnCHT3A, and miR-305-5p can specifically inhibit the transcription of MnCHT3A in vivo.