The aim of this study was to evaluate the effect of the co-administration of vitamin D and omega-3 fatty acid on clinical, metabolic and genetic parameters in women with polycystic ovary syndrome (PCOS).This randomized, double-blinded, placebo-controlled clinical trial was conducted on 60 subjects, aged 18–40 years old with PCOS. Subjects were randomly allocated to take either 50,000 IU vitamin D every 2 weeks plus 2000 mg/day omega-3 fatty acid from fish oil (n = 30) or placebo (n = 30) for 12 weeks. Gene expression analysis of inflammatory cytokines was conducted on peripheral blood mononuclear cells (PBMCs) of PCOS women using RT-PCR method.Vitamin D and omega -3 fatty acid co-supplementation significantly decreased serum total testosterone levels (−0.2 ± 0.5 vs. + 0.1 ± 0.4 ng/mL, P = 0.02) compared with the placebo. In addition, vitamin D and omega-3 fatty acid co-supplementation resulted in a significant improvement in beck depression inventory (−1.4 ± 1.6 vs. −0.5 ± 0.6, P = 0.01), general health questionnaire scores (−4.5 ± 4.3 vs. −1.9 ± 2.3, P = 0.005) and depression anxiety and stress scale scores (−5.0 ± 5.1 vs. −2.3 ± 3.5, P = 0.01) compared with the placebo. Additionally, vitamin D and omega-3 fatty acid co-administration significantly decreased serum high-sensitivity C-reactive protein (hs-CRP) (−1.2 ± 1.9 vs. + 0.1 ± 0.7 mg/L, P = 0.001) and malondialdehyde (MDA) levels (−0.4 ± 0.4 vs. + 0.2 ± 0.6 µmol/L, P < 0.001), and significantly increased plasma total antioxidant capacity (TAC) levels (+ 114.6 ± 122.2 vs. -2.4 ± 168.2 mmol/L, P = 0.003) compared with the placebo. Results of RT-PCR demonstrated that vitamin D and omega-3 fatty acid co-supplementation significantly downregulated gene expression of interleukin-1 (IL-1) (P = 0.03), and upregulated vascular endothelial growth factor (VEGF) (P = 0.004) in PBMCs of subjects with PCOS, when compared with placebo.Overall, the co-administration of vitamin D and omega-3 fatty acid for 12 weeks had beneficial effects on mental health parameters, serum total testosterone, hs-CRP, plasma TAC and MDA levels, and gene expression of IL-1 and VEGF among women with PCOS.