Threonine (Thr) is one of the most limiting amino acids in functional ingredients used for diet preparation, and its deficiency can reduce fish growth. This study aimed to estimate the digestible Thr requirement in tambaqui (Colossoma macropomum) diets in the fattening stage for 60 days. Diets containing different digestible Thr concentrations (9.40, 10.30, 11.20, 12.10 and 13.00 g/kg) were analysed. The best results for growth performance, body crude protein content and body deposition occurred in fish fed with 10.30 and 11.20 g Thr kg diet −1 (p < 0.05). The dietary Thr requirement estimated based on weight gain, the feed conversion ratio and the specific growth rate was 10.64, 10.71 and 10.80 g Thr kg diet −1 respectively (p < 0.05). For body deposition, the best values of dietary Thr were between 10.40 and 10.70 g/kg (p < 0.05). Diets with higher levels of digestible Thr caused an increase in plasma cholesterol, liver glycogen levels and erythrocytes, and a decrease in hepatic aspartate aminotransferase levels and mean corpuscular volume (p < 0.05). In conclusion, tambaqui require between 10.40 and 10.80 g Thr kg diet −1 to provide better fish growth performance, body deposition and health.