Background: Human follicular fluid (FF) is rich in hormones and antioxidants. Many components of FF differ in follicles of patients with polycystic ovary syndrome (PCOS). Regarding vitamin D effects on gene expression, 25(OH)D level of FF and its association with oxidative status and sex steroids dysregulation in PCOS group was evaluated and compared to controls of Non-obese healthy women. Methods: FF of 50 non-obese healthy women and 50 women with PCOS (18-36 years old) who were candidates for IVF/ICSI was aspirated on the oocyte retrieval day. Sex steroids and 25(OH)D levels were measured by ELISA. Reactive oxygen species (ROS) levels, total antioxidant capacity (TAC), and activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were assessed by chemiluminescence and spectrophotometric methods. Data were analyzed by unpaired t-test or Mann-Whitney test, and Pearson correlation coefficient. The p<0.05 was considered statistically significant. Results: Estradiol, progesterone, 25(OH)D, TAC, and activities of SOD, GPx, and CAT in FF of women with PCOS were significantly lower, whilst their free and total testosterone and ROS levels were significantly higher than controls. There were significant positive correlations between FF levels of 25(OH)D with TAC, estradiol and progesterone concentrations, SOD, GPx, and CAT activities. Negative correlations were found between 25(OH)D with free and total testosterone, and ROS levels. Conclusion: Despite different hormonal and antioxidant levels in FF of normal and cystic follicles, the correlation between 25(OH)D levels with sex steroids and oxidative stress markers showed a possible role of 25(OH)D in regulating sex hormones secretion and enhancement of antioxidant defense.