Background: Polycystic ovary syndrome (PCOS) is the most challenging endocrinopathy in reproductive-age women owing to the continuous necessity for treatment modifications to satisfy the patient's needs. Gut dysbiosis and vitamin D deficiency have been found in women with PCOS, and their modulation is a target for medical research. So, the study was done to estimate the potential effects of both vitamin D3, probiotics, and co- administration in a letrozole-induced PCOS model in female albino rats. Methods: Forty-five female albino rats were separated into five equal groups: control, supplemented with vehicle orally daily till the end of the study, PCOS; given letrozole 1mg/kg BW orally daily for 21 days for PCOS induction then given the vehicle daily for 28 days, probiotics-treated PCOS; supplemented with the probiotic (Linex Probio-Tec AB Blend 64) 210 mg/kg/day for 28 days orally, D3-treated PCOS; supplemented with vitamin D3 (1000 IU/kg/day) for 28 days orally and D3+Probiotics-treated PCOS; supplemented with both vitamin D3 and probiotics for 28 days. All treatments started after induction of PCOS by letrozole for 21 days. Blood samples and ovarian tissues were taken at the end of the study for biochemical and histopathological investigations. Results: Treatment with either probiotics or vitamin D3 improved the ovarian polycystic morphological and hormonal changes and enhanced the serum adiponectin, lipid profile, glycemic, inflammatory, and oxidative changes compared with PCOS rats. The combined-treated PCOS group showed a remarkable synergistic effect on ameliorating all the studied parameters compared with either treated group alone. Conclusions: Both probiotics and vitamin D3 had beneficial therapeutic effects in the letrozole-induced rat model of PCOS. Co-administration of probiotics and vitamin D3 had synergistic effects on ameliorating the polycystic ovarian changes and provided a promising therapeutic line for treating the PCOS