Background: The widely recognized health benefits of physical activity have led to the development of programs designed to increase activity levels within workplaces, where inactivity is often prevalent. We aimed to evaluate how participation in a Scotland-wide workplace walking program (Step Count Challenge [SCC]) influenced participants’ physical fitness and mental well-being. Methods: A 3-part multistudy design was employed, each using the 2-minute step test and Short Warwick-Edinburgh Mental Well-Being Scale to measure physical fitness and mental well-being, respectively. Study 1 (Spring, 8 wk, N = 475) and study 2 (Autumn, 4 wk, N = 336) were repeated-measures online-based nationwide cohort studies in 2021, and study 3 (N = 38) was an in-person experimental design with a control group in 2023. Results: Studies 1 and 2 found significant improvement in physical fitness (Spring: P < .001, η2 = .19; Autumn: P = .014, η2 = .13) and mental well-being (Spring: P < .001, η2 = .13; Autumn: P = .007, η2 = .05). Study 3 found a significant group × time interaction for physical fitness (P = .018, η2 = .172) such that the SCC group significantly improved over time (P = .042, η2 = .131) and found no significant differences for mental well-being. Conclusions: The findings of studies 1 and 2 demonstrate that participation in SCC is associated with a positive effect on physical fitness and mental well-being regardless of seasonal and duration differences. Study 3 also showed improvement in physical fitness, with a significantly greater increase for those who participated in SCC. This supports the promotion of future workplace walking programs.