Aim: Psychosocial factors in the working environment have been shown to be associated with mobility limitations, but this has not yet been confirmed in a Danish population. We aimed to examine how psychosocial factors at work are related to developing mobility limitations in Denmark. Methods: This study is based on data from 2952 middle-aged men and women without mobility limitations in 2000. Results: We found increased risk of incident mobility limitations during 6-year follow up among men who often perceived high work pace (OR 5.45, 95% CI 1.21–24.52) vs. never, who only sometimes or/never perceived the work to be meaningful (OR 6.54, 95% CI 1.55–27.55) vs. always, and who sometimes perceived high emotional demands at work (OR 7.85, 95% CI 1.78–34.65) vs. never. Among women, lower risk of incident mobility limitations was observed among those who in 2000 perceived high work pace sometimes (OR 0.46, 95% CI 0.24–0.87) or often (OR 0.43, 95% CI 0.22–0.85) vs. never in 2000. Also, women who always or often experienced high emotional demands had an increased risk. Conclusions: The most important finding was that high work pace was strongly associated with increased risk of mobility limitations among men, but associated with lower risk of mobility limitations among women. This knowledge may be used to better target interventions among men and women in midlife from physical deterioration later in life.