Abstract: This study investigates the effects of different dietary riboflavin levels on the mitochondrial biogenesis and function of oriental river prawn (Macrobrachium nipponense) average initial weight: (0.67±0.01) g). Three semi-purified diets with different riboflavin concentrations (3.91, 169.61, and 326.66 mg/kg) were tested on prawns kept in an indoor recirculating water system for 10 weeks. The three treatments were designated as the deficient, optimal, and over-dosed groups, respectively. The results showed that in the 169.61 mg/kg riboflavin group, feed efficiency, hepatopancreatic Na⁺-K⁺-ATPase and mitochondrial complex III activities, cytochrome C content, and Cytochrome C oxidase-Ⅱ (cox-Ⅱ) transcription were all significantly higher (P<0.05) than those in the 3.91 and 326.66 mg/kg groups. The hepatopancreatic ATP content and transcriptions of cox-Ⅰ and ATP synthase in the 169.61 mg/kg riboflavin group were all significantly higher than those of the 3.91 mg/kg group (P<0.05), but showed no significant difference with the 326.66 mg/kg group (P>0.05). However, the average daily gain, hepatopancreatic succinate dehydrogenase activity in the 169.61 mg/kg riboflavin group was significantly higher than that in the 326.66 mg/kg group (P<0.05), with no significant difference compared to the 3.91 mg/kg group (P>0.05). Additionally, the 326.66 mg/kg group showed significantly higher citrate synthase activity (P<0.05) than the other groups and a higher mitochondrial complex V activity than the 3.91 mg/kg group (P<0.05). Furthermore, the AMP/ATP ratio in the 3.91 mg/kg group was significantly higher compared with the other groups (P<0.05). These findings suggest that dietary inclusion of 169.61 mg/kg riboflavin can promote mitochondrial biogenesis and function in M. nipponense.