Abstract Background Prolonged sitting has been negatively associated with a range of non-communicably diseases. However, the role of occupational sitting is less clear, and little is known on the changes of occupational sitting in a working population over time. The present study aimed to determine 1) temporal changes in occupational sitting time between 1990 and 2010 in the Danish workforce; 2) the association and possible dose-response relationship between occupational sitting time and all-cause mortality. Methods This study analysed data from the Danish Work Environment Cohort Study (DWECS), which is a cohort study of the Danish working population conducted in five yearly intervals between 1990 and 2010. Occupational sitting time is self-reported in the DWECS. To determine the association with all-cause mortality, the DWECS was linked to the Danish Register of Causes of Death via the Central Person Register. Results Between 1990 and 2010 the proportion of the Danish workforce who sat for at least three quarters of their work time gradually increased from 33.1 to 39.1 %. All-cause mortality analyses were performed with 149,773 person-years of observation and an average follow-up of 12.61 years, during which 533 deaths were registered. None of the presented analyses found a statistically significant association between occupational sitting time and all-cause mortality. The hazard ratio for all-cause mortality was 0.97 (95 % CI: 0.79; 1.18) when ≥24 hr/wk occupational sitting time was compared to