Background: Vitamin D plays a key role in maintaining skeletal health, but is also related to various non-skeletal health issues. Several determinants have been identified that influence blood plasma levels of 25-hydroxyvitamin D (25(OH) D), often in specific patients or elderly populations. This paper aims to replicate these findings in a healthy population. Methods: Plasma levels of 25(OH)D were measured using tandem mass spectrometry. We examined the cross-sectional association of sociodemographic, health, lifestyle and sampling characteristics with 25(OH)D in a group of 539 adults, who were healthy control subjects in the NESDA study in the Netherlands (latitude 52 °N). Results: Mean 25(OH)D levels were 68.0 (±27.2) nmol/l. Levels under 50 nmol/l occurred in 27% of the population; 40% reached levels above 75 nmol/l. Women had higher levels than men, and the use of oral contraceptives showed a significant positive association among females. Subjects with non-European ancestry had dramatically lower 25(OH) D levels. Other factors that were negatively associated were body mass index and the renal estimated glomerular filtration rate (eGFR). Meteorological data replaced season as a significant determinant. Moderate alcohol consumption and sports showed a positive association, while physical activity and the hepatic marker gamma-glutamyl transferase did not. Our results disconfirm the influence of age in this population of under 65 year olds. Conclusion: Insufficient 25(OH)D levels were common in a healthy population. The set of eight variables that were significant in a multiple regression model (sex, ancestry, oral contraceptives, eGFR, BMI, sports, alcohol, sunshine) explained 29.5% of the variance.