Introduction: The purpose of this study was to compare fat and carbohydrate oxidation at different intensities of exercise until exhaustion, between professional and elite amateur cyclists. Materials and Methods: Forty-two professional cyclists (27.3± 4.2 yr) and 31 elite amateur cyclists (23.7±3.6yr) performed incremental exercise tests on a bicycle ergometer with 50 Watt increments, every third minutes until volitional exhaustion. Heart rate, the average volume of oxygen consumption, production of carbon dioxide and respiratory exchange ratio were measured during the rest time and the last 3 minutes of the exercise with working loads of 200, 300 and 400 Watts. Results: The results showed that there was no significant difference in RER between the two groups at rest time and intensity of 200w, but at intensities of 300w and 400w, RER was significantly lower in professional cyclists (P=0.01). There was no significant difference between professional and elite amateur cyclists regarding the rate of fat and carbohydrate oxidation at different intensities of exercise. Also, there was no significant difference in the percentage of fat and CHO oxidation during the trials, between the two groups. With increasing exercise intensity, the fat oxidation rate decreased significantly (p=0.001) however between rest mode and intensity of 200w, the difference was not significant. Conclusion: The findings of present study suggest that although there is a little difference in fat and carbohydrate oxidation between professional and elite amateur cyclists, this small difference and the small amount of carbohydrates saved in professional cyclists leads to their superior performance.