Both fatigue and negative work-life balance can be influenced by job characteristics and individual differences, while fatigue is associated with reduced positive well-being. This paper reports a study that investigated the mediation effect of fatigue between those stressors and well-being outcomes among UK railway staff. A large number of significant mediation effects of fatigue were found in this study, and as a result, the process by which job demands, job support and control influence major positive well-being outcomes can be partially explained by fatigue.