Background: This study investigated the therapeutic effects of Rosa canina L. seed oil (RCSO) on PCOS using an animal model, focusing on evaluating biochemical factors and the Bax/Bcl-2/p53 and Caspase-3 (Cas-3) pathways. Materials and Methods: The PCOS model was created by injecting estradiol valerate into 60 adult Wistar rats, which were assigned to six groups of 10 animals: normal, PCOS, metformin, RCSO, and co-treatment with MET and RCSO groups. After 50 days, the serum levels of testosterone, progesterone, LH, and FSH hormones were measured, along with the activity of catalase, superoxide dismutase, glutathione peroxidase, and nitric oxide. Additionally, ovarian tissues were taken for gene expression analysis of Bax/Bcl-2/p53 and Cas-3 using real-time PCR, as well as protein expression analysis with western blot. Immunohistochemistry was performed to assess the levels of p53 and Ki-67 proteins, indicating the apoptosis of ovarian granulosa cells. Results: RCSO, in synergy with MET, demonstrated reductions in the LH/FSH ratio along with a decrease in progesterone and increased activity of antioxidant enzymes. Additionally, RCSO, along with MET, upregulated the Bax/Bcl-2/p53 and Cas-3 pathways, resulting in increased p53 expression and decreased Ki-67-positive cells, indicative of enhanced apoptosis. Conclusions: These findings suggest the potential use of RCSO in women with ovulation disorders, particularly those with PCOS.