Vitamin D (Vit D) deficiency has been linked to symptoms of polycystic ovary syndrome (PCOS), yet little is known about Vit D supplementation as a treatment for sexual dysfunction (SDy) in women with PCOS. To explore the implications of serum total 25-hydroxyvitamin D (25[OH]D) and bioavailable 25[OH]D (bio-25[OH]D) status and replacement on women with PCOS and SDy. Reproductive-age women with PCOS who were not desiring fertility were eligible provided that they also had SDy, as assessed by the Female Sexual Function Index (FSFI), and were without severe depression, as evaluated by the Beck Depression Inventory II (BDI-II). Participants were given the recommended dietary allowance of Vit D (600IU daily) plus hormonal contraception (HC; cyclic ethinyl estradiol/drospirenone) or no HC for 6months. Comparisons between groups were analyzed by chi-square test and t-test, and Pearson's correlation coefficient analyzed correlations between FSFI with demographics, BDI-II, androgen levels, and total and bio-25[OH]D. The outcomes included SDy (FSFI <26.55), total and serum bio-25[OH]D levels, and total and free testosterone. A total of 42 women without severe depression completed the FSFI, with 28 (66.7%) having SDy. All FSFI domains, including arousal, lubrication, orgasm, and pain, were significantly lower as compared with women without SDy, with no associations with respect to demographics, total and free testosterone, or total and bio-25[OH]D. Vit D replacement was initiated with HC (n = 18) or no HC (n = 10), and for those completing the study, FSFI improved (score >26.55) in 61% (11/18) regardless of the treatment group. A time-treatment effect showed a significant change for the domain of orgasm, suggesting that HC had more of an impact than Vit D replacement. Improvement in sexual function as a dichotomous variable was not associated with age, body mass index, other demographics, total and free testosterone, total and bio-25[OH]D, or HC use. Due to the prevalence of SDy in women with PCOS, efficacious treatment options are necessary. This study is the first to analyze the effect of Vit D supplementation on SDy in women with PCOS. Limitations included the small number of participants who completed the study, thus limiting meaningful conclusions and generalizability. Vit D status was not associated with SDy and BDI-II. While HC may have played a role, standard Vit D supplementation could not account for the noted improvement in FSFI in women with PCOS.