Introduction: Brain-Derived Neurotrophic Factor (BDNF), besides its major role in behavioral disorders, plays an important role in controlling food intake and weight. The combined effects of exercise and diet on BDNF levels, have not been completely identified. Therefore, the current study was aimed to investigate the effect of acute aerobic exercise following different diets, on serum levels of BDNF and Cortisol in overweight men. Materials and Methods: In this quasi-experimental crossover study, 10 overweight men (23±6.1 years old and 29.26±0.47 BMI), attended our physiology lab, in 4 sessions with one week intervals. In each session, after consuming Normal (N), High-carbohydrate (HCHO), High-protein (HP) and High-fat (HF) diets, the subjects performed a 30 min treadmill aerobic exercise, with 60-70% of maximum heart rate, and furthermore, blood samples were taken to measure serum levels of BDNF and Cortisol at fasting, 2 hour after the diet (before exercise), and after exercise. Results: Serum levels of BDNF showed a significant increase after acute aerobic exercise, following HCHO (P=0.001), HP (P=0.001) and N (P=0.039) diets compared to the HF diet. There was no significant difference in serum levels of cortisol after aerobic exercise, following the 4 different diets (P=0.258). Conclusion: It seems that calorie intake from the HF diet, reduces serum levels of BDNF in overweight men, and aerobic exercise after consuming this diet, despite the lack of changes in Cortisol levels, does not increase BDNF levels.