Background <p>This explorative study investigated the associations between working hours, biological aging, and all-cause and cardiovascular disease (CVD) mortality, and assessed the potential mediating role of lifestyle factors.</p> Methods <p>We utilized data from the Older Finnish Twin Cohort, which comprised 9246 participants who had reported their weekly working hours in 1990. Biological aging was assessed using blood-based epigenetic aging measures in a subsample (<i>n</i> = 740). We used a counterfactual mediation approach. Lifestyle-related factors, including leisure-time physical activity (LTPA), alcohol consumption, body mass index (BMI), smoking, and sleep, were considered potential mediators. Between-within twin models were used to evaluate the extent to which shared familial factors confounded the observed associations.</p> Results <p>During the mean 28-year follow-up period, 20.5% of the participants died. Long hours were directly associated with decreased risk of all-cause mortality, but only at the between-pair level; there were no associations with biological aging. However, long hours were indirectly associated with an increased risk of all-cause and CVD mortality through a higher BMI and smoking, and with an increased risk of CVD mortality through lower LTPA levels. Long hours were also indirectly associated with accelerated biological aging through smoking. Part-time work was indirectly associated with a lower risk of all-cause and CVD mortality through a lower BMI.</p> Conclusions <p>The findings of this explorative study suggest that direct associations between long working hours, biological aging, and mortality are shaped by lifestyle-mediated pathways and shared familial factors.</p>