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Sperm mitochondrion is one of the major susceptible organelles that get damaged during cryopreservation. The study aimed to minimize mitochondrial dysfunction and oxidative stress during sperm cryopreservation using mitochondria‐specific antioxidants. For this, semen was collected from five buffalo bulls (3 ejaculates/bull). The ejaculates were diluted in an low‐density lipoprotein‐based extender and divided into four equal aliquots. Mitochondria‐targeted antioxidant (MitoQ) was added at a final concentration of 0 (control), 0.02, 0.2 and 2 μM separately in each aliquotes and cryopreserved. The addition of MitoQ at a concentration of 0.02 μM improved post‐thaw sperm motility, plasma membrane integrity and able to sustain sperm motility for a longer time. To investigate MitoQ's effects on mitochondrial function, we measured mitochondrial membrane potential (MMP) using JC‐1 dye, superoxide production …