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Background: Polycystic ovary syndrome (PCOS) is a set of reproductive and metabolic symptoms associated with an imbalance of reproductive hormones in women. Macrophage migration inhibitory factor (MIF) is an inflammatory adipocytokine associated with metabolic disorders. Probiotics probably has influence on metabolic and inflammatory conditions. We aimed by this study, for the first time, to evaluate the impact of probiotic supplementation on MIF values, clinical and laboratory feature of PCOS. Furthermore, we aimed to explore the power of MIF for diagnosis PCOS. Methods: Sixty women diagnosed with PCOS were selected in this single-blinded clinical trial and 40 healthy control women. Participants received probiotic supplements for 12-week. Clinical and laboratory measures were evaluated at baseline and after 12 weeks of intervention. High sensitivity C-reactive protein (hs-CRP) and MIF levels were assessed using an enzyme-linked immunosorbent assay (ELISA). Results: Probiotic supplementation improved clinical, hormonal, and inflammatory biomarkers in women with PCOS. Additionally in PCOS group, logistic regression analysis revealed that MIF and HOMA-IR were independent predictors of probiotic response with odds ratios of 1.641 and 0.335, respectively. Regarding inflammatory biomarkers, hs-CRP and MIF, its value increased in PCOS group compared to healthy group. Receiver operating characteristic curve (ROC) analysis revealed that, area under the curve (AUC) for the MIF was 0.981 (95% CI = 0.902–0.898) with sensitivity = 90.2%, specificity = 89.8% and the cutoff values (19.7). Interestingly, the power of MIF was more sensitive and specific than hs-CRP for diagnosis of PCOS. Thus MIF could be a useful diagnostic inflammatory biomarker discriminating PCOS from healthy controls. Moreover in PCOS group stepwise multiple linear regression analysis, detected that HOMA-IR, neutrophil count, hs-CRP and fat mass index (FMI) were main predictors of MIF levels among other clinical and laboratory biomarkers. Conclusion: Regarding the effects of probiotic supplementation on inflammatory and metabolic disorders; our novel findings explored a favorable effect of probiotic supplementation on inflammatory biomarkers; clinical, laboratory features of PCOS, and MIF levels. Furthermore, probiotic could be used for treatment of PCOS and its associated morbidity.