Introduction: Train driving is a high responsibility job in railway industry. Train drivers need different cognitive functions such as vigilance, object detection, memory, planning, decision-making. High level of fatigue is one of the caused factor of accidents among train drivers. Numerous factors can impact train drivers’ fatigue but high level of workload is a key factor. Therefore, the aim of the present study was to investigate workload and its relationship with fatigue among train drivers in Keshesh section of Iranian Railway Company. .Material and Method: This descriptive analytical study was done among 100 train drivers in Keshesh section of Iranian Railway industry. They were selected by simple random sampling. The NASA-TLX workload scale and Samn-Perelli fatigue scale were respectively used to investigate workload and fatigue. Data were analyzed by Paired t-test and Spearman correlation coefficient. . Result: According to the NASA-TLX results, effort and mental workload with the mean score of 74/22 and 73/31 were respectively the most important attributes of workload among train drivers. No significant relationship was observed between workload and level of fatigue before departure and half an hour before reaching the destination station (P>0.05). However, the relationship between of workload and level of fatigue half an hour before the end of shift (on the way back to the origin station) was statistically significant (P=0.048) among the sample population. . Conclusion: Effort and mental workload were the most important attributes of workload among train drivers. By focusing on these two variables and adopting fatigue management programs, fatigue and workload can be controlled and the efficiency of the whole system can be enhanced accordingly.