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Vitamin D deficiency was associated with obesity. But their causal relationship is still controversial. CYP27B1 plays a direct role in circulating the concentration of 25(OH)D, which can be used to investigate causal relationships. In this study, allele T of rs10877012 and allele T of rs4646536 in CYP27B1 were family-based linked with both vitamin D deficiency and obesity. In addition, both rs10877012 and rs4646536 were associated with serum 25(OH)D3 levels between siblings but not BMI. Therefore, vitamin D deficiency may be a causal factor of obesity. Maintaining sufficient vitamin D is beneficial to obesity prevention.