Background: Although sperm cryopreservation seems to be an efficient technique for distributing competent sperm for artificial insemination, the process affects the quality of post-thawed sperm.Objectives: This study was designed to see how the novel mitochondria-targeted antioxidant “Mito-TEMPO” affected buck sperm quality during cryopreservation. Methods: After proper semen samples collection, they were diluted and divided into 5 equal groups and cryopreserved in liquid nitrogen with 0, 1, 10, 100, and 1000 µM Mito-TEMPO. Sperm motility, lipid peroxidation, abnormal morphology, acrosome integrity, membrane integrity, and viability were all evaluated after thawing. Results: When the freezing extender was supplemented with 10 µM Mito-TEMPO, total motility, progressive motility, membrane integrity, acrosome integrity, and viability increased (P≤0.05), while lipid peroxidation decreased (P≤0.05). Conclusion: Finally, the novel mitochondria-targeted antioxidant “Mito-TEMPO” could be introduced as an effective cryo-additive to improve buck semen quality parameters during cryopreservation.