This research aimed to improve the post-thaw quality of ram semen by reducing oxidative stress during the freezing process through the use of Ganoderma lucidum (Reishi), a mushroom known for its potent antioxidant properties. Semen samples collected from 6 Akkaraman rams were pooled and divided equally into control and treatment groups. The study consists of six groups: Group 1 (Control, 0% GL), Group 2 (0.5% GL), Group 3 (1% GL), Group 4 (2% GL), Group 5 (4% GL), Group 6 (8% GL). After dilution and cooling, the semen was glycerolized, equilibrated, packaged into 0.25 ml mini straws, and cryopreserved using an automated freezing system. Results showed that supplementation with 0.5%, 1%, 2% GL significantly improved total and progressive motility compared with the control group. In contrast, the 8% GL treatment exhibited toxic effects on sperm, leading to reduced motility. In the kinematic analysis, significant changes were detected in velocity of curvilinear and velocity of average path values of 8% GL compared to all other groups. In the oxidative stress analysis, malondialdehyde concentrations were significantly higher in the 8% GL group compared with the control group. Glutathione concentrations and catalase activity increased in the 1% and 2% GL groups and decreased in the 8% GL group. Glutathione peroxidase activity increased in the 0.5%, 1% and 2% groups and decreased in 8%. Superoxide dismutase activity decreased in the 8% GL group but remained unchanged in the other treatment groups. Overall, the findings suggest that supplementation of extenders with 0.5%, 1%, or 2% GL extract enhances semen cryopreservation quality in rams.