Riboflavin as an indispensable water-soluble B vitamin, is particularly crucial for the growth and metabolism of animals. It often participates in biochemical reactions as flavin adenine dinucleotide (FAD) and flavin mononucleotides (FMN), and plays a key role in the normal growth and the development of animal body. To date, the research data on the appropriate vitamin requirements of oriental river prawn (Macrobrachium nipponense) are relatively lacking compared to other nutrients, which has slowed the process of research and development of formula feed, as well as hindered the efficient and economic development of aquaculture. Therefore, this experiment was conducted to investigate the effects of different dietary riboflavin levels on the growth performance and metabolic function of M. nipponense, in order to help the construction of the nutritional requirements database of this species, and facilitate the development of high quality formula feed. A total of 1200 oriental river prawns with an average weight of 0.68 g were picked out, and randomly separated into 6 groups, with 4 replicates in each group, and 50 prawns in each tank, respectively. Six semi-purified diets were prepared, containing 3.91, 20.53, 49.18, 87.80, 169.61 and 326.66 mg/kg of riboflavin. Subsequently, a 10-week feeding trial was conducted in an indoor recirculation water system. The results showed that: ① the 169.61 mg/kg group had significantly higher final weight, weight gain rate, specific growth rate, protein retention rate, plasma pyruvate level, and hepatopancreatic riboflavin content compared with other groups, while the feed intake, feed conversion ratio, and plasma urea nitrogen levels were significantly lower than the other groups. Using the weight gain rate and hepatopancreatic riboflavin content as evaluation indicators to establish the regression models, it was indicated that the optimal riboflavin requirement of M. nipponense was 165.25-180.31 mg/kg; ② the mRNA levels of glucose transporter 2, hexokinase, pyruvate kinase, hormone sensitive lipase, glutamate dehydrogenase, and glutamine synthase all increased significantly with the increase of dietary riboflavin levels, and maximized in the 169.61 mg/kg group, then decreased; the mRNA levels of pyruvate carboxylase, fatty acid synthase, and acetyl CoA carboxylase all showed an opposite result, and minimized in the 169.61 mg/kg group. In summary, adding 165.25-180.31 mg/kg of riboflavin significantly promoted the growth performance and feed utilization of M. nipponense, and enhanced the glycolysis, fatty acids β-oxidation, and protein synthesis pathways. This study preliminarily clarified the molecular mechanism of riboflavin to improve the growth performance and metabolic function of M. nipponense, which can provide technical guidance for the construction of nutrition demand database of M. nipponense and the research and development of efficient formula feed.