Little is known about the role of dietary amino acids on male reproductive performance and gamete quality in fishes. Thus, the objective of this study was to investigate how “enhanced” feeds (EH- 4, EH-5, EH-6), with modified amino acid composition, and the standard on-growing diet (DAN-EX) impact body composition, milt biochemistry, and sperm performance in male European eel, Anguilla anguilla. The fatty acid composition of EH-4, EH-5, and EH-6 was similar but differed to that in DAN-EX, while amino acid composition varied between all four diets. Diet did not influence organ-somatic indices (e.g. HSI, GSI), while males fed EH-4 were heavier than other groups. Arginine, alanine, and lysine were the most abundant amino acids in milt (>11%), followed by glycine, aspartic acid, valine, glutamic acid, and leucine (>5.66%). Diet mpacted milt arginine, serine, proline, methionine, and histidine levels. Specifically, males fed DAN-EX, EH-4, and EH-5 had the highest percentages of arginine, while males fed EH-4 to EH-6 had higher percentages of serine. Proline was most abundant in males fed DAN-EX, EH-5, and EH-6. Both methionine and histidine were detected at low percentages (<2%), and were impacted by diet, where males fed EH-4 and EH-5 had higher percentages of methionine, and males fed DAN-EX, EH-4, and EH-6 had the highest percentage of histidine. Milt production increased over time, where eels fed EH-4 and EH-6 showed the highest probability of producing suited milt volumes (>0.5 mL) for fertilization procedures. Spermatocrit (43.1 ± 1.80%) did not differ between the diets (ranged from 37.57 to 47.21%). Dietary regime had an impact on sperm motility, such that eels fed EH-5 and EH-6 had the greatest percentage of motile cells. In addition, fish fed EH-5 and EH-6 (or DAN-EX) had the fastest swimming sperm. Spermatogenic maturity index of hormonally treated eels varied within groups but did not differ between dietary treatment groups after 9 weeks of injections (ranged from 0.54 to 0.80). The most interesting amino acids to scrutinize from PCA plots were proline, histidine, and valine as well as lysine and arginine. Here, eels with highly motile sperm had milt with high relative proportions of proline, histidine, and valine, but were particularly low in lysine and arginine. Together, our findings add evidence that certain amino acids regulate milt biochemistry, and that male ejaculate traits may be promoted by amino acid intake. Further studies to evaluate effects of supplemented amino acid diets on fertilization ability and inter-linked early developmental stages are required.