Cryopreservation in rooster semen is a helpful procedure to spread qualified semen samples for reproductive goals. Nevertheless, some post-thawed qualified semen samples showed a considerably poor fertility rate that might be related to epigenetic modifications during the cryopreservation process. This study aims to investigate the effect of the cryopreservation process in the presence of MitoQ as a mitochondrial-targeted antioxidant on epigenetic changes and other quality parameters (motility, morphology, mitochondrial activity, acrosome integrity, lipid peroxidation, DNA fragmentation, apoptosis status, and ROS concentration) of rooster sperm. The collected semen samples were divided into four groups of fresh samples and three groups that were supplemented by MitoQ 0, 10, and 100 nM and cryopreserved. The cryopreservation process reduced (p ≤ 0.05) DNA methylation, H3K9 acetylation, H3K4 methylation, motility parameters, membrane integrity, mitochondrial activity, acrosome integrity, viability, and increased (p ≤ 0.05) lipid peroxidation, DNA fragmentation, ROS concentration, and apoptotic-like changes compared to the fresh semen group. However, in frozen sperm groups, MitoQ 10 and 100 nM resulted in significant improvements (p ≤ 0.05) in the epigenetic modifications and other mentioned quality parameters compared to the control group (MitoQ 0). Generally, although the cryopreservation process reduced semen quality, using MitoQ could be useful in cryopreserved rooster semen quality.