Objectives: The purpose of this research was to evaluate the relationship between overtime work hours and CAVI, a new index of arterial stiffness. Methods: We measured CAVI of Japanese workers (3,862 men) aged 26 to 59 years. Simultaneously, we obtained information on their monthly overtime work hours for the past few months using a self-administered questionnaire, with responses divided into five groups: <45, ≥45 and <60, ≥60 and <80, ≥80 and <100 and ≥100 hours/month. We calculated the odds ratios of CAVI≥9.0 for each group of overtime work hours. Results: In the full sample, there was no significant association between the average CAVI and overtime work hours. Taking<45 overtime hours/month as the reference category, the odds ratios of CAVI≥9.0 were as follows: OR=1.11, 95% CI=0.73–1.69 (≥45 and <60 hours/month); OR=0.92, 95% CI=0.48–1.76 (≥60 and <80 hours/month); OR=1.50, 95% CI=0.50–4.49 (≥80 and <100 hours/month); and OR=2.65, 95% CI=0.82–8.54 (≥100 hours/month). However, for workers in their 50 s, the odds ratio of CAVI≥9.0 was significantly higher among subjects with≥100 hours/month than among those with<45 hours/month (OR=4.26, 95% CI=1.2–15.1) Conclusions: The present study suggests that CAVI is more likely to be 9.0 or higher in workers in their 50 s when they work≥100 hours of overtime per month.(J Occup Health 2014; 56: 271-278)