Simple SummaryThe rapid gonad reproduction of hatchlings restricts the sustainable development ofMacrobrachium nipponense. Thus, it is urgently required to establish an artificial technique to regulate the process of gonad reproduction inM. nipponense. A previous study predicted that Cyclin B3(CycB3) may perform crucial functions in the regulation of male reproduction inM. nipponense. In the present study, we aimed to investigate the potential roles of CycB3 in the male reproduction of this species. qPCR analysis results suggested that CycB3 was involved in the process of spermiogenesis, oogenesis, and embryogenesis inM. nipponense. RNA interference analysis showed that CycB3 affected the expression of insulin-like androgenic gland hormone and inhibited testis reproduction inM. nipponense. Taken together, these findings suggest that CycB3 plays essential roles in the regulation of male reproduction inM. nipponense, promoting the studies of the regulation of testis reproduction.AbstractCyclin B3 (CycB3) is involved in the metabolic pathway of the cell cycle, playing essential roles in the regulation of cell proliferation and mitosis.CycB3is also predicted to be involved in the reproduction of male oriental river prawns (Macrobrachium nipponense). In this study, the potential functions ofCycB3inM. nipponensewere investigated using quantitative real-time PCR, RNA interference, and histological observations. The full-length DNA sequence ofCycB3inM. nipponensewas 2147 base pairs (bp) long. An open reading frame of 1500 bp was found, encoding 499 amino acids. A highly conserved destruction box and two conserved cyclin motifs were found in the protein sequence ofMn-CycB3. Phylogenetic tree analysis revealed that this protein sequence was evolutionarily close to that ofCycB3s of crustacean species. Quantitative real-time PCR analysis results suggested thatCycB3was involved in the process of spermiogenesis, oogenesis, and embryogenesis inM. nipponense. RNA interference analysis showed thatCycB3had a positive regulatory relationship with insulin-like androgenic gland hormone (IAG) inM. nipponense. In addition, sperm were rarely observed in the testis ofdouble-stranded CycB3-injected prawns after 14 days of treatment, and sperm abundance was dramatically lower than that in thedouble-stranded GFP-injected prawns on the same day. This result indicated thatCycB3can regulate the testis reproduction inM. nipponensethrough inhibiting theIAGexpressions. Overall, these results indicated thatCycB3plays essential roles in the regulation of male reproduction inM. nipponense, which may promote the studies of male reproduction in other crustacean species.