Individuals who are physically fit or engage in regular physical activity have a lower incidence of cardiovascular disease and risk of mortality. We conducted a large-scale controlled trial of interventions to decrease cardiovascular risk factors, during which we assessed the effect of a workplace-based intervention program, which was part of a population strategy for promoting long-term increases in physical activity, on the blood lipid profiles of participating employees. Data were collected from 2929 participants and this report presents the results of a survey conducted in five factories for the intervention group and five factories for the control group at baseline and year 5. The absolute/proportional changes in HDL-cholesterol were 2.7 mg/dL (4.8%) in the intervention group and -0.6 mg/dL (-1.0%) in the control group. The differences between the two groups in the change in serum levels of HDL-cholesterol were highly significant (p<0.001) in each analysis of covariance, in which the number of cigarettes smoked was included or excluded. In the intervention group, the daily walking time increased significantly (p<0.001) when compared between baseline and year 5, whereas no significant difference was observed in daily walking time in the control group over the identical period. Our results show that an intervention program promoting physical activity raises serum HDL-cholesterol levels of middle-aged employees. Increased awareness of the benefits of physical activity, using environmental rearrangement and health promotion campaigns, which especially target walking, may have contributed to a beneficial change in serum HDL-cholesterol levels in the participants.