Abstract: This study evaluated the effects of dietary supplementation with Bacillus megaterium on growth performance, accumulation of phosphorus and calcium, and intestinal health of juvenile American eels (Anguilla rostrata). A total of 480 juvenile A. rostrata with an initial body weight of (14.00±0.03) g were randomly divided into 4 groups (BM0, BM1.6, BM3.2, and BM6.4), each with four replicates, and fed a basal diet supplemented with 1.6×10⁸, 3.2×10⁸, and 6.4×10⁸ CFU/g B. megaterium for 56 days. Compared with the BM0 group, the growth performance in BM1.6 and BM3.2 groups increased significantly (P<0.05), and no significant difference was observed between the BM6.4 and BM0 groups (P>0.05). The levels of calcium and phosphorus in whole fish and the accumulation rates of dietary calcium and phosphorus were significantly increased in B. megaterium supplemental groups. The levels of serum calcium, phosphorus, and complement 3 were significantly increased in the BM supplementation group (P<0.05); and content of serum 1, 25-dihydroxy vitamin D_3, levels of parathyroid hormone, and immunoglobulin M, along with activity of lysozyme were significantly higher in the BM1.6 and BM3.2 groups (P<0.05), with no significant differences between BM0 and BM6.4 groups (P>0.05). The intestinal protease and lipase activities were higher than those of BM0 group (P<0.05). The intestinal total antioxidant capacity level, glutathione peroxidase, catalase, and superoxide dismutase activities were significantly increased (P<0.05), and malondialdehyde level was significantly decreased (P<0.05) in the BM1.6 and BM3.2 groups compared to the BM0 group, no significant differences were found between the BM0 and BM6.4 groups (P>0.05). Compared with BM0 group, the villi length and muscular thickness of the intestine were significantly increased in the BM1.6 and BM3.2 groups (P<0.05), and no significant difference between the BM0 group and BM6.4 group (P>0.05). At the phylum level, the relative abundances of Proteobacteria and Cyanobacteria decreased in the BM1.6 group but increased in the BM6.4 group, whereas Firmicutes and Actinobacteria increased in BM1.6 but decreased in BM6.4. The relative abundances of Bacteroidota increased in the BM1.6 and BM6.4 groups. At the genus level, the relative abundance of Escherichia-Shigella was higher in the BM0 group than that in the BM1.6 and BM6.4 groups (P<0.05), the relative abundance of Lysinibacillus in BM1.6 was higher than that in BM0 and BM6.4 groups (P<0.05), and Acinetobacter, Acidovorax, Herbaspirillum, Aquabacterium, and Ralstonia in BM6.4 were higher than those in BM0 and BM1.6 groups (P<0.05). In conclusion, appropriate dietary supplementation with B. megaterium can significantly improve growth, increase calcium and phosphorus accumulation in whole fish, and enhance intestinal health of juvenile A. rostrata. The recommended supplementation level is 1.6×10⁸-3.2×10⁸ CFU/g of diet.