Background: Branched-chain amino acids (BCAAs, isoleucine, leucine, and valine) are commonly applied to promote muscle protein synthesis. However, the effects of BCAAs on exercise-induced substrate metabolism, performance and post-exercise fatigue during endurance exercise remain unclear. Methods: In a double-blind cross-over design, eleven active males completed 1 h of constant load exercise (CLE) at 60% VO₂max power followed by a time to exhaustion (TTE) test at 80% VO₂max power after supplementation with BCAAs or placebo on consecutive three days. During exercise, indirect calorimetry was used to measure the carbohydrate (CHO) and fat oxidation rate, as well as the cycling efficiency. In addition, rating of perceived exertion (RPE) and visual analogue scale (VAS) scores were obtained at interval times during the whole period. Fingertips and venous blood (n = 8) were collected for the measurement of metabolic responses at different time points during exercise. Results: Compared to the placebo group, the fat oxidation rate was significantly higher after 20 and 30 min of CLE (p < 0.05). The CHO oxidation rates showed a significant increase in the BCAA group during TTE (p < 0.05). Meanwhile, the cycling efficiency during TTE was significantly improved (p < 0.05). Interestingly, VAS significantly decreased post-exercise in the BCAA group (p < 0.05). Additionally, the levels of blood insulin between the two groups were significantly higher in the post-exercise period compared to the pre-exercise periods (p < 0.001), while insulin levels were significantly lower in the post-exercise period with supplemental BCAAs compared to the placebo (p < 0.001). BCAAs also enhanced the levels of blood ammonia in the post-exercise period compared to the fasting and pre-exercise periods (BCAA: p < 0.01; Placebo: p < 0.001). However, in the post-exercise period, blood ammonia levels were significantly lower in the BCAA group than in the placebo group (p < 0.05). Conclusions: This study shows the critical role of BCAAs during exercise in active males and finds that BCAA supplementation enhanced fat oxidation during the CLE, increased carbohydrate oxidation and exercise efficiency during the TTE, and reduced immediate post-exercise fatigue.