Abstract: Purpose: We studied the effects of a physical activity and dietary intervention on plasma lipids in a general population of children. We also investigated how lifestyle changes contributed to the intervention effects. Methods: We carried out a 2-year controlled, non-randomized lifestyle intervention study among 504 mainly prepubertal children aged 6–9 years at baseline. We assigned 306 children to the intervention group and 198 children to the control group. We assessed plasma concentrations of total, LDL, HDL, and VLDL cholesterol, triglycerides, HDL triglycerides, and VLDL triglycerides. We evaluated the consumption of foods using 4-day food records and physical activity using a movement and heart rate sensor. We analyzed data using linear mixed-effect models adjusted for age at baseline, sex, and pubertal stage at both time points. Furthermore, specific lifestyle variables were entered in these models. Results: Plasma LDL cholesterol decreased in the intervention group but did not change in the control group ( − 0.05 vs. 0.00 mmol/L, regression coefficient (β) = − 0.0385, p = 0.040 for group*time interaction). This effect was mainly explained by the changes in the consumption of high-fat vegetable oil-based spreads (β = − 0.0203, + 47% change in β) and butter-based spreads (β = − 0.0294, + 30% change in β), moderate-to-vigorous physical activity (β = − 0.0268, + 30% change in β), light physical activity (β = − 0.0274, + 29% change in β) and sedentary time (β = − 0.0270, + 30% change in β). The intervention had no effect on other plasma lipids. Conclusion: Lifestyle intervention resulted a small decrease in plasma LDL cholesterol concentration in children. The effect was explained by changes in quality and quantity of dietary fat and physical activity. Clinical Trial Registry Number: NCT01803776, ClinicalTrials.gov