The rising cost of fish meal (FM) in aquaculture feed has prompted the search for alternative protein sources like soybean meal (SBM). However, SBM-based diets can negatively affect fish growth, lipid metabolism, and intestinal health. One possible solution is to add cholesterol to SBM-based feeds to mitigate these adverse effects, but the optimal levels and the resulting benefits remain unclear. In this study, the impact of adding cholesterol to low FM and high soybean protein diets on the growth, lipid metabolism, and intestinal health of Monopterus albus was evaluated. Juvenile M. albus (initial body weight of 20.00 +/- 0.02 g) were fed a control diet with 42% FM + 22% SBM diet (FM group), 22% FM + 52% SBM diet (SBM group), and the SBM diet supplemented with 5 g/kg or 10 g/kg cholesterol (SBC5 and SBC10 groups, respectively). The results showed that the weight gain rate (WGR) and hepatosomatic index (HSI) were significantly lower in the SBM group compared to the FM group, but WGR increased with the addition of 10 g/kg cholesterol. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities increased significantly in the SBM group, indicating liver stress, but returned to normal levels in the SBC10 group. Cholesterol supplementation also improved serum and liver lipid profiles and significantly increased the contents of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in serum and high-density lipoprotein cholesterol (HDL-C) in the liver. In addition, cholesterol supplementation increased the activities of intestinal enzymes (e.g., amylase) and restored the structural integrity of the intestinal lining, including villus height and goblet cell count. Additionally, the SBC10 group exhibited a richer and more diverse intestinal microbiota, with increased abundance of Firmicutes and Bacteroidota. These findings demonstrate that supplementing 10 g/kg cholesterol in a high SBM diet improves growth performance, lipid metabolism, intestinal health, and gut microbiota composition in M. albus. This study provides a practical food-borne strategy to enhance the use of plant-based proteins in aquaculture while mitigating their negative effects, contributing to the sustainability of fish farming.