ObjectiveThe aim of this study was to determine the effect of vitamin D and probiotic co-administration on mental health, hormonal, inflammatory and oxidative stress parameters in women with polycystic ovary syndrome (PCOS).MethodsThis randomized, double-blinded, placebo-controlled clinical trial was carried out on 60 subjects, aged 18–40 years old. Subjects were randomly allocated to take either 50,000 IU vitamin D every 2 weeks plus 8 × 109 CFU/day probiotic (n = 30) or placebo (n = 30) for 12 weeks.ResultsVitamin D and probiotic co-supplementation, compared with the placebo, significantly improved beck depression inventory [β (difference in the mean of outcomes measures between treatment groups) − 0.58; 95% CI, − 1.15, − 0.02; P = 0.04], general health questionnaire scores (β − 0.93; 95% CI, − 1.78, − 0.08; P = 0.03) and depression, anxiety and stress scale scores (β − 0.90; 95% CI, − 1.67, − 0.13; P = 0.02). Vitamin D and probiotic co-supplementation was associated with a significant reduction in total testosterone (β − 0.19 ng/mL; 95% CI, − 0.28, − 0.10; P < 0.001), hirsutism (β − 0.95; 95% CI, − 1.39, − 0.51; P < 0.001), high-sensitivity C-reactive protein (hs-CRP) (β − 0.67 mg/L; 95% CI, − 0.97, − 0.38; P < 0.001) and malondialdehyde (MDA) levels (β − 0.25 μmol/L; 95% CI, − 0.40, − 0.10; P = 0.001), and a significant increase in total antioxidant capacity (TAC) (β 82.81 mmol/L; 95% CI, 42.86, 122.75; P < 0.001) and total glutathione (GSH) levels (β 40.42 μmol/L; 95% CI, 4.69, 76.19; P = 0.02), compared with the placebo.ConclusionsOverall, the co-administration of vitamin D and probiotic for 12 weeks to women with PCOS had beneficial effects on mental health parameters, serum total testosterone, hirsutism, hs-CRP, plasma TAC, GSH and MDA levels.Trial RegistrationThis study was retrospectively registered in the Iranian website (www.irct.ir) for registration of clinical trials (IRCT20170513033941N37).