© 2019, The Japan Diabetes Society. Aim: Understanding work-related factors associated with regular exercise in employees could be useful for determining appropriate interventions to prevent the onset and aggravation of diabetes. This study aimed to assess the associations among work-related stress and mental fatigue, and regular exercise in Japanese employees with or without diabetes. Methods: This cross-sectional study was conducted in 2018, and included 2916 full-time employees aged older than 40 years without cardiovascular disease, cancer, respiratory disease, kidney disease, disc herniation, and depression. Work-related stress was measured by the Job Content Questionnaire and work-related mental fatigue was measured using the Numerical Rating Scale (NRS). Results: Regular exercise was present in 23.9% of 117 employees with diabetes and in 21.3% of 2799 employees without diabetes. In employees with diabetes, the multivariable-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for regular exercise were 0.26 (0.07–0.96) in those with an NRS score of 4–6 (moderate) and 0.22 (0.06–0.85) in those with an NRS score of 7–10 (severe) compared with those with an NRS score of 0–3 (none or mild), after adjusting for confounding factors. Similarly, in employees without diabetes, the multivariable-adjusted ORs (95% CIs) for regular exercise were 0.77 (0.62–0.97) in those with an NRS score of 4–6 and 0.75 (0.59–0.94) in those with an NRS score of 7–10 compared with those with an NRS score of 0–3. Conclusions: Work-related mental fatigue was negatively associated with regular exercise in employees with and without diabetes.