Abstract: In order to evaluate the effects of different levels of pancreatin (from porcine pancreas) on the growth, digestion, liver and intestinal health of juvenile largemouth bass (Micropterus salmoides), a study was conducted where 40% of fish meal protein was replaced with cottonseed meal. Pancreatin was added at levels of 250, 500, 750, 1000, and 1250 mg/kg levels (TG-250, TG-500, TG-750, TG-1000 and TG-1250, respectively), ensuring isonitrogenous and isolipidous conditions. A normal commercial diet served as the negative control group (NCG), and the cottonseed meal replacement (without pancreatin) group served as the positive control group (PCG). The juvenile largemouth bass initial body weight (8.00±0.50) g was fed for 70d. The results showed that replacing fish meal with cottonseed meal decreased feed utilization rate, growth, and digestive performance (P<0.05), and adversely affected liver and intestinal health (P<0.05). However, adding pancreatin significantly increased final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR), and survival rate (SR), and decreased the feed coefficient (P<0.05) in the range of 250—750 mg/kg. At 750 mg/kg, the condition factor (CF) increased (P<0.05), while the visceral index (VSI) and hepatosomatic index (HSI) decreased (P<0.05), and crude protein content increased (P<0.05). The activities of protease, amylase, and lipase in the stomach, foregut, and liver increased in the range of 250—1000 mg/kg (P<0.05). At 750 mg/kg pancreatin, the apparent digestibility of dry matter, crude protein, and crude fiber was the highest, and the nitrogen and phosphorus content in feces was the lowest. In the range of 250—750 mg/kg, malondialdehyde (MDA) levels decreased (P<0.05), while catalase (CAT), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities increased (P<0.05). At 750 mg/kg, the relative expression of sod and cat gene in liver was up-regulated (P<0.05), the relative expression of anti-inflammatory factors IL-10 and TGF-β was the highest, while the relative expression of pro-inflammatory factors IL-1β, IL-8, and TNF-a was the lowest. Additionally, the expressions of occludin, zo-1, and claudin-1 in the intestinal tract was up-regulated (P<0.05), and the activities of diamine oxidase (DAO), D-lactic acid (D-Lac), and endotoxin (ET) in serum were decreased (P<0.05). In conclusion, the replacement of 40% fish meal protein by cottonseed meal had a significant effect on the growth and health of juvenile largemouth bass. The growth performance, digestive enzyme activity, and apparent digestibility could be significantly improved by adding appropriate amount of pancreatin (750 mg/kg), as well as the liver and intestinal health. Therefore, pancreatin can be used as a high-quality additive for juvenile largemouth bass feed.