Objective：To investigate the association between vitamin D(VD) deficiency and the pathogenesis of polycystic ovarian syndrome (PCOS). Methods : Vitamin D levels were detected in 34 patients with PCOS and 30 healthy women, and the correlation analysis was performed. Vitamin D deficiency mouse model and PCOS mouse model were constructed and divided into four groups：Control group (CTR group), PCOS group, Vitamin D deficiency group (VD- group), and Vitamin D deficiency combined with PCOS group (VD-+PCOS group). Estrous cycle and ovary morphological changes were observed. Serum sex hormone indexes and glucose lipid metabolism indexes were detected. Real?time quantitative PCR(RT-qPCR) was used to detect the micro RNA (mRNA) levels of hormone synthase and sex hormone receptor in ovarian tissues. Results: The levels of serum 25-hydroxyvitamin D[25(OH)D] in PCOS women were significantly lower than those in normal healthy women[(16.49±6.50)ng/mL vs (20.08±5.28)ng/mL, P=0.019].The levels of luteinizing hormone(LH),testosterone (TT),LH/FSH ratio and free androgen index(FAI) in PCOS group were significantly higher than those in control group, while the levels of sex hormone binding globulin (SHBG) were significantly lower than those in control group, and the difference were statistically significant (r<0，P<0.05). Correlation analysis showed that serum 25(OH)D levels of the total sample population were negatively correlated with LH(r=-0.2713,P<0.05),LH/FSH ratio(r=-0.3137,P<0.05),TT(r=-0.2757,P<0.05),prolactin[PRL,(r=-0.2741,P<0.05)] and FAI (r=-0.3118,P<0.05),and serum 25(OH)D levels in PCOS patients were negatively correlated with PRL(r=-0.4042,P<0.05).Vitamin D deficiency stalled the estrous cycle in mice. Compared with the CTR group, the serum LH levels of mice in other groups were significantly increased (P<0.05).Compared with the CTR group, the mRNA level of androgen receptor (AR) in ovarian tissues of mice in VD- group was increased (P<0.05). Conclusion: Vitamin D deficiency existed in patients with PCOS. There was a negative correlation between serum 25(OH)D and PRL in PCOS patients. Vitamin D deficiency disturbs normal estrous cycle and affects sex hormone levels in mice. Vitamin D deficiency may participate in the occurrence of PCOS by regulating the expression of hormone synthesis-related genes.