Abstract: This study aimed to investigate the effects of supplementation with different ratios of fish oil to soybean oil on the growth performance and lipid metabolism of juvenile American eels (Anguilla rostrata), and to determine the appropriate ratio for such supplementation in the provided diet. A total of 600 juvenile eels (initial body weight: 11.34±0.01 g) were randomly assigned to five treatment groups, each fed a basal diet supplemented with additional fish oil and soybean oilat ratios of 10:0 (F10S0), 7﹕3 (F7S3), 5﹕5 (F5S5), 3﹕7 (F3S7), or 0﹕10 (F0S10). The total additional supplementation level of fish oil and soybean oil in the diet was 5% for each group. Each treatment comprised four replicates of 30 fish per replicate, and the trial lasted 70 days. Compared with the F10S0 group, the F5S5 group exhibited significantly improved growth performance (P<0.05). No significant differences in body composition were observed among groups (P>0.05). However, the F5S5 group showed significantly lower serum total cholesterol (P<0.05). Intestinal lipase, amylase, and protease activities were significantly elevated in the F7S3, F5S5, and F3S7 groups (P<0.05), with the highest digestive enzyme activities observed in the F5S5 group. Hepatic lipid droplet accumulation was significantly reduced in the F5S5 group (P<0.05), whereas the F3S7 and F0S10 groups exhibited markedly increased lipid droplet numbers (P<0.05). Furthermore, the F5S5 group displayed significantly decreased hepatic lipogenic capacity and increased lipolytic capacity (P<0.05). In summary, supplementation with an appropriate ratio of fish oil and soybean oil promotes growth performance, elevates intestinal digestive enzyme activities, reduces serum lipid levels, inhibits hepatic lipogenesis, augments lipolysis, and decreases hepatic lipid accumulation in juvenile American eels. A 5﹕5 ratio of fish oil and soybean oil is recommended for this supplementation, and the results serve as a reference for the rational inclusion of oils in eel feed.