Background Workplace health promotion (WHP) has been proposed as a preventive intervention for job stress, possibly operating by promoting positive organizational culture or via programs promoting healthy lifestyles. The aim of this study was to investigate whether job stress changed over time in association with the availability of, and/or participation in a comprehensive WHP program (Healthy@Work). Method This observational study was conducted in a diverse public sector organization (~28,000 employees). Using a repeated cross-sectional design with models corroborated using a cohort of repeat responders, self-report survey data were collected via a 40 % employee population random sample in 2010 (N = 3406) and 2013 (N = 3228). Outcomes assessed were effort and reward (self-esteem) components of the effort-reward imbalance (ERI) measure of job stress. Exposures were availability of, and participation in, comprehensive WHP. Linear mixed models and Poisson regression were used, with analyses stratified by sex and weighted for non-response. Results Higher WHP availability was positively associated with higher perceived self-esteem among women. Women’s mean reward scores increased over time but were not statistically different (p > 0.05) after 3 years. For men, higher WHP participation was associated with lower perceived effort. Men’s mean ERI increased over time. Results were supported in the cohort group. Conclusions For women, comprehensive WHP availability contributed to a sense of organizational support, potentially impacting the esteem component of reward. Men with higher WHP participation also benefitted but gains were modest over time and may have been hindered by other work environment factors.