Background <p>The current study was conducted to assess the effect of probiotic and vitamin D co-supplementation on migraine index (MI), migraine-specific quality of life (MSQoL), and oxidative stress in adults with migraine.</p> Methods <p>This parallel randomized, triple-blinded, placebo-controlled trial was conducted among adult individuals aged 18 to 55 years with a diagnosis of migraine headache according to the International Classification of Headache Disorders-3 (ICHD-3). Patients were randomized to either multispecies probiotic (4.5 × 10¹¹ CFU per day) plus vitamin D (50,000 IU every two weeks) or placebo for 12 weeks. MI, MSQoL, blood pressure, anthropometric, and biochemical variables (25-hydroxy vitamin D, nitric oxide (NO), malondialdehyde (MDA), total oxidative status (TOS), total antioxidant capacity (TAC), and catalase (CAT)) were examined at baseline and after 12 weeks of intervention.</p> Results <p>In total, 72 patients (36 patients in each group) with migraine headache were included in the study. The mean age of patients was 37.46 ± 0.98 and sixty five out of 72 patients were females. A significant increase in mean serum levels of 25-hydroxy vitamin D was observed in the probiotic plus vitamin D group compared to the placebo group (<i>p</i> &lt; 0.001). A significantly greater reduction in mean MI (-30.11 ± 6.95 vs. -11.97 ± 3.05; <i>p</i> = 0.01) was found in the probiotic plus vitamin D than in the placebo group. In addition, a marginally significant difference was observed between the two groups regarding changes in serum levels of NO (<i>p</i> = 0.05).</p> Conclusion <p>This trial showed that probiotic plus vitamin D supplementation can favorably improve MI as well as serum levels of NO in adult patients with migraine. Further research on mechanisms through which the supplementation was effective on MI is warranted.</p>