We aimed to examine the association between exposure to work stress and chronic disease incidence and loss of chronic disease-free life years in the Danish workforce. The study population included 1,592,491 employees, aged 30–59 in 2000 and without prevalent chronic diseases. We assessed work stress as the combination of job strain and effort-reward imbalance using job exposure matrices. We used Cox regressions to estimate risk of incident hospital-diagnoses or death of chronic diseases (i.e., type 2 diabetes, coronary heart disease, stroke, cancer, asthma, chronic obstructive pulmonary disease, heart failure, and dementia) during 18 years of follow-up and calculated corresponding chronic disease-free life expectancy from age 30 to age 75. Individuals working in occupations with high prevalence of work stress had a higher risk of incident chronic disease compared to those in occupations with low prevalence of work stress (women: HR 1.04 (95% CI 1.02–1.05), men: HR 1.12 (95% CI 1.11–1.14)). The corresponding loss in chronic disease-free life expectancy was 0.25 (95% CI − 0.10 to 0.60) and 0.84 (95% CI 0.56–1.11) years in women and men, respectively. Additional adjustment for health behaviours attenuated these associations among men. We conclude that men working in high-stress occupations have a small loss of years lived without chronic disease compared to men working in low-stress occupations. This finding appeared to be partially attributable to harmful health behaviours. In women, high work stress indicated a very small and statistically non-significant loss of years lived without chronic disease.