Vitamin D is associated with established cardiovascular risk factors such as low density lipoprotein (LDL) in adults. It is unknown whether these associations are present in early childhood. To determine whether serum 25-hydroxyvitamin D (25(OH)D) is associated with serum non-high density lipoprotein (non-HDL) cholesterol during early childhood we conducted a cross-sectional study of children aged 1 to 5 years. Healthy children were recruited through the TARGet Kids! practice based research network from 2008-2011 (n=1,961). The associations between 25(OH)D and non-fasting non-HDL cholesterol (the primary endpoint), total cholesterol, triglycerides, HDL, and low density lipoprotein (LDL) cholesterol, were evaluated using multiple linear regression adjusted for age, sex, skin pigmentation, milk intake, vitamin D supplementation, season, body mass index, outdoor play, and screen time. Each 10 nmol/L increase in 25(OH)D was associated with a decrease in non-HDL cholesterol concentration of -0.89 mg/dl (95% CI: -1.16,-0.50), total cholesterol of -1.08 mg/dl (95%CI: -1.49,-0.70), and triglycerides of -2.34 mg/dl (95%CI: -3.23,-1.45). The associations between 25(OH)D and LDL and HDL were not statistically significant. 25(OH)D concentrations were inversely associated with circulating lipids in early childhood, suggesting that vitamin D exposure in early life may be an early modifiable risk factor for cardiovascular disease.