Abstract: The mud crab (Scylla paramamosain) is the most aquacultured marine crab species in China, and its aquaculture industry has enormous economic benefits. However, mud crab aquaculture currently relies mainly on biological baits, and the lack of high-quality artificial pellet diet has restricted the sustainable development of this industry. The basis for the research and development of artificial pellet diet is to clarify the requirements for basic nutrients. Essential amino acids are amino acids that cannot be synthesized by the body or are synthesized at an insufficient rate and must be provided by food. The content of essential amino acids in diet directly affects the growth, metabolism and physiological functions of aquacultured animals. To identify the valine demand of juvenile S. paramamosain (the initial weight was 7.90±0.90 mg), this study conducted a 50-day feeding trial using five experimental diets with different valine levels (1.10%-2.28%). The results showed that the dietary valine levels significantly affected the growth performance, body composition, digestive and transaminase activities, antioxidant capacity and hepatopancreatic bacterial community structure of S. paramamosain. Dietary containing 1.58%-1.85% valine significantly increased the final body weight (FBW), weight gain (WG), specific growth rate (SGR) of S. paramamosain, as well as increased the crude protein and crude lipid levels of the whole-body. Meanwhile, the appropriate dietary valine levels (1.58%-1.85%) increased the amylase and alanine aminotransferase activities of S. paramamosain. The dietary incorporation of 1.58% and 1.85% valine significantly increased the transcription levels of genes related to the PI3K/AKT/mTOR signaling pathway. In addition, the lowest hepatopancreatic malondialdehyde (MDA) content, the highest superoxide dismutase (SOD) activity and the total antioxidant capacity (T-AOC) were obtained in the 1.85% dietary valine level group of S. paramamosain. On the other hand, the present study found that appropriate dietary valine levels maintained hepatopancreatic bacterial flora health by increasing the abundance of probiotic bacteria (unclassified_f_Rhodobacteraceae, Ruegeria, Paracoccus, Leisingera, and Ilumatobacter) and inhibiting the abundance of pathogenic bacteria (Vibrio, unclassified_f_Flavobacteriaceae, and unclassified_f_Weeksellaceae). In summary, the second-order polynomial and double broken line model analyses based on WG concluded that the valine demand of juvenile S. paramamosain was 1.63%-1.76%. The results of this study improve the nutritional requirement database of S. paramamosain and provide basic data for the development of high-quality artificial compound diets for S. paramamosain.