Abstract Objective To investigate associations between employees' health locus of control (HLOC) and self-perceived health, health behaviors, and participation in health promotion programs (HPPs) and the mediating effect of self-perceived health and health behaviors on the relation between HLOC and participation. Method Between 2010 and 2012, a six-month longitudinal study was conducted among 691 Dutch employees. Using questionnaires, information was collected on health behaviors, self-perceived health, HLOC, and intention to participate at baseline. Actual participation was assessed at follow-up. Logistic regression analyses were used to study associations between HLOC and self-perceived health, health behaviors, and participation, and to examine whether associations between HLOC and participation were mediated by self-perceived health and health behaviors. Results Higher internal HLOC was associated with sufficient physical activity (moderate: OR:1.04, 95%CI:1.00–1.08; vigorous: OR:1.05, 95%CI:1.01–1.10) and fruit and vegetable intake (OR:1.05, 95%CI:1.01–1.09), a good self-perceived health (OR:1.20, 95%CI:1.11–1.30), a positive intention towards participation (OR:1.05, 95%CI:1.00–1.09), and actual participation (OR:1.06, 95%CI:1.00–1.13). Self-perceived health or health behaviors did not mediate associations between HLOC and participation. Conclusion Employees with a higher internal HLOC behaved healthier and were more likely to participate in HPPs, irrespectively of their health. Increasing internal HLOC seems a promising avenue for improving employees' health and participation in HPPs.