CONTEXT: Factors associated with the high prevalence of vitamin D deficiency in China are not well described, especially among Chinese adolescents. OBJECTIVES: The aim of the study was to examine important environmental or sociodemographic factors influencing 25-hydroxyvitamin D [25(OH)D] levels and estimate its heritability. DESIGN: A sample of 226 male and female adolescent twins aged 13-20 yr from a large prospective twin cohort of rural Chinese children and adolescents that has been followed for 6 yr were evaluated. MAIN OUTCOME MEASURE(S): Blood level of 25(OH)D was measured using tandem mass spectrometry methodology. RESULTS: The overall mean (SD) 25(OH)D level was 18.0 (9.4) ng/ml, with wide variation by gender and season. In males (47.4% of subjects), the mean (SD) 25(OH)D level was 12.1 (4.2) ng/ml in non-summer and 27.4 (8.8) ng/ml in summer; in females, it was 10.1 (4.1) ng/ml in non-summer and 19.5 (6.3) ng/ml in summer. A multivariate model that included gender, age, season, physical activity, and student status demonstrated that male gender, summer season, and high physical activity significantly increased 25(OH)D levels. Summer season and male gender also significantly decreased the risk of being in the lowest 25(OH)D tertile. Overall, 68.9% of the variability in 25(OH)D level was attributable to additive genetic influence. Stratification by gender found that in males, 85.9% of the variability in 25(OH)D level was attributable to such influence, but in females, it was only 17%. CONCLUSION: In this sample of rural Chinese adolescents, 25(OH)D level was influenced by gender, season, and physical activity level. There was a strong genetic influence on 25(OH)D level in males only.