This study aimed to determine the dietary lysine requirements of juvenile Colossoma macropomum tambaqui based on growth performance. We also evaluated gut and hepatic histomorphometry as well as blood metabolites in accordance with the increased levels of dietary lysine. The juveniles (33.88 ± 2.47 g) were fed until apparent satiation with diets containing 6.60, 9.72, 12.84, 15.96, 19.08 and 22.20 g/kg of lysine. Fish were randomly distributed in groups of 10 fish per tank and assays were performed in triplicate, during 90 days. Tambaqui fed with 15.96 g/kg dietary lysine showed higher final weight (p = 0.001) and optimized feed conversion ratio (p = 0.001). Morphohistological modifications were present in livers of fish fed with low levels of lysine. In the proximal intestine, mucosa layer density was greater at the level of 15.96 g/kg (p = 0.001). In the middle intestine, height (p = 0.001) and perimeter (p = 0.001) of the villi were greater at low levels of lysine (respectively, 9.72 and 12.84 g/kg dietary lysine). Tambaqui fed with 15.96 g/kg of lysine achieved higher plasma protein concentrations (p = 0.01). Using the second-order polynomial regression analysis as support, and based on protein efficiency rate and body weight gain, dietary lysine requirement for juvenile tambaqui was calculated as 15.4–15.6 g/kg of diet (5.7–5.8% of dietary protein).