Background: Polycystic ovary syndrome (PCOS) is the commonest challenging endocrinopathy in reproductive age women. Gut dysbiosis and deficiency of vitamin D have been found in women with PCOS and their modulation is a target for medicinal research. So, the study was done to estimate the potential effects of vitamin D3, probiotics and co-administration of both in letrozole-induced rat model of PCOS. Methods: Forty five female albino rats were separated into 5 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 then given the vehicle daily for 28 days, probiotics-treated; supplemented with the probiotics 210 mg/kg/day for 28 days orally, D3-treated; supplemented with vitamin D3 (1000 IU/kg/day) for 28 days orally and D3+Probiotics-treated; 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, hormonal and biochemical parameters compared with PCOS rats. Combined-treated group showed a remarkable synergistic effect on ameliorating all the studied parameters compared with either treated groups alone. Conclusion: Both probiotics and vitamin D3 have beneficial therapeutic effects in letrozole-induced rat model of PCOS. Co-administration of probiotics and vitamin D3 has synergistic effects on ameliorating the polycystic ovarian changes and provide a promising therapeutic line for treating the PCOS.