This study was conducted to assess the influence of supplementing Tris-extender with Coenzyme Q10 (COQ10) as an antioxidant on sperm characteristics of Egyptian buffalo and cattle. This study was carried out at Animal Production Research Station, El-Gemmezah, belonging to Animal Production Research Institute, Egypt. Five sexually mature buffalo (4 y and 400-450 kg) and Friesian (3 y and 350-400 kg) bulls were used in this study. Semen was collected twice weekly for five successive weeks. Only ejaculates with ≥70% mass motility were pooled and diluted at 37°C with Tris-extender containing 0, 20 and 30 μM of CoQ10 in T1, T2 and T3, respectively. Equilibration period was at 5°C for 4 h and semen was packed in 0.25 ml French straws frozen in liquid nitrogen -196^oC. Stored straws were thawed individually at 37°C for 30 s. Semen was evaluated for sperm characteristics including percentages of progressive motility, livability, abnormality, curled tail and acrosomal damage of spermatozoa. Enzymatic activity of AST, ALT and LDH was determined in seminal plasma of post-thawed semen. Results showed that there were no species differences in all sperm characteristics studied, except for increasing (P<0.05) percentage of abnormality and damage acrosome spermatozoa in post-thawed semen of cattle compared with buffalo bulls. All sperm characteristics were improved (P<0.05) by 30 μM COQ10 supplementation T3 in post-diluted, post-equilibrated and post thawed semen as compared to control without supplementation T1 in buffalo and cattle semen. Activity of all enzymes reduced (P<0.05) in seminal plasma of both species. In conclusion, CoQ10, as antioxidant, at a level of 30 μM in Tris-extender is able to enhance most sperm characteristics including mobility, livability, plasma membrane integrity, with decreasing sperm abnormalities and it has strong protective power against acrosomal damage of buffalo and cattle spermatozoa.