The present study was conducted to determine the effect of reduced glutathione (GSH) and superoxide dismutase (SOD) supplementation in the bull semen freezing extender on post-thaw parameters of Holstein and Simmental bull sperm. Semen were collected from seven bulls (four Holstein and three Simmental) and cryopreserved in the Tris-egg-yolk based extender as control group and supplemented with various concentrations of GSH × SOD (5 × 100, 7.5 × 100, 5 × 150, and 7.5 × 150 mM × IU mL(-1)) in treatment groups. Microscopic parameters were evaluated in terms of total motility parameters using computer assisted semen analysis and viability and membrane integrity were assessed using Eosin-Nigrosin stains and hypo-osmotic swelling test (HOST), respectively after thawing the semen. Malonaldialdehyde (MDA) level, SOD and glutathione peroxides (GPx) activities were assessed immediately after thawing. Results showed that supplementation of the cryopreservation medium with various concentrations of GSH × SOD improved total motility (TM) and progressive motility parameters for Holstein (P < 0.05) semen, and values of TM and HOST for Simmental semen compared to the control group (P < 0.01) after semen thawing. Addition of antioxidant to Holstein semen samples decreased the level of MDA and increased GPx activities compared to control groups (P < 0.05). SOD activities increased in Simmental bull samples compare to the control group (P < 0.01), but not differ in Holstein, while these activities. In conclusion, supplementation of antioxidant to the semen extender as combination (GSH × SOD) improved the semen post-thaw qualities which may be associated with a reduction in lipid peroxidation as well as an increase in the antioxidant enzyme activities.