Abstract: This study aimed to investigate the effects of dietary vitamin A and determine its requirement of juvenile red swamp crayfish (Procambarus clarkii). Six isonitrogenous, isolipidic, and isoenergetic experimental diets were formulated with analyzed vitamin A potencies of 11573 (A1), 20042 (A2), 26274 (A3), 37818 (A4), 55159 (A5), and 79200 (A6) IU/kg, respectively. These diets were fed to crayfish with an initial average body weight of (3.40±0.01) g for 8 weeks. The results revealed that weight gain rate and specific growth rate were significantly higher in groups A4 and A5 compared to the control group (A1), (P<0.05), with the highest values observed in group A4. In contrast, feed conversion ratio and hepatosomatic index were significantly decreased (P<0.05), reaching the lowest values in group A4. Compared to the control group, the total antioxidant capacity, catalase, and superoxide dismutase activities in the hepatopancreas were significantly enhanced (P<0.05) in group A4. Serum aspartate aminotransferase and alanine aminotransferase activities decreased initially and then increased with rising vitamin A levels, both attaining their minimum in group A4. Simultaneously, the incidence of B-cells and R-cells in the hepatopancreas, intestinal villi length and width, and activities of hepatopancreatic trypsin, amylase, lipase, and intestinal trypsin were significantly elevated in group A4 (P<0.05). Furthermore, group A4 significantly up-regulated the gene expression levels of Chitinase, EcR, and RXR in the hepatopancreas, while significantly down-regulating the expression of MIH (P<0.05). In conclusion, optimal vitamin A supplementation improve digestive function, enhance hepatopancreas antioxidant and immune capacity, and regulate ecdysis-related gene expression, thereby promoting growth performance in P. clarkii. Based on broken-line regression analysis of weight gain rate, specific growth rate, and feed conversion ratio, the recommended dietary vitamin A requirement for juvenile Procambarus clarkii is 37976—43443 IU/kg.