Cryopreservation induces sub lethal damage to the spermatozoa, thereby reduce their fertile life. In the present study, effect of glutathione on bovine semen freezability and in vitro fertilizing potentials were evaluated. Semen was cryopreserved in tris-based extender supplemented with different concentrations of reduced glutathione (0, 0.5, 1, 2, 3 and 5 mM). Semen post-thawing motility, viability and acrosomal integrity, DNA damage, enzymes leakage, total antioxidant capacity (TAC), lipid peroxidation and in vitro fertilizing potentials were assessed. Current results indicated that addition of 2 mM glutathione to semen extender significantly (P<0.05) improved post-thawing motility, viability and acrosomal integrity (66.25±5.54%, 169.38±18.59and 12.75±3.45%, respectively) compared with control (45.00 ±2.89%, 95.00±8.90 and 23.75±3.45 %, respectively). Likewise, at this concentration sperm DNA damage, tail length and tail moment of the cryopreserved semen significantly (P<0.05) reduced (1.54±0.38%, 1.58±0.19µm and 2.95±1.01, respectively) compared with control (6.07±1.61 %, 6.12±1.16 µm and 34.61±6.32, respectively). Moreover, 2 mM glutathione significantly (P<0.05) increased TAC (0.50±0.07 mµ/ml) and decreased lipid peroxidation of the cryopreserved spermatozoa (9.68±2.72 nmol/ml) with respect to the control (0.19±0.01 mµ/ml, and 24.82±4.90 nmol/ml, respectively). Additionally, 2 mM glutathione significantly (P<0.05) improved in vitro fertilization rate, cleavage rate and development to morula and blastocyst stages (56.78±12.85, 50.18±6.88, 24.68±5.75 and 17.66±3.19%, respectively) compared with the control (28.10±2.21, 27.01±4.15, 8.80±10.4 and 3.90±2.31%, respectively). It was concluded that the addition of 2 mM glutathione to the freezing extender improved freezability and enhanced in vitro fertilizing potentials of bovine spermatozoa through protection of DNA from deterioration and reduction of oxidative stress.