This study was conducted to investigate the effect of semen extenders enriched with glutathione (GSH) on in vitro quality parameters and fertility of post-thawed turkey. In experiment 1, pools of semen diluted in glucose-based extender containing 0.5, 1, and 2 mM of GSH were cryopreserved. During the next step, a different variable such as motility and motion parameters, plasma membrane integrity (PMI) and functionality (PMF), DNA integrity, lipid peroxidation (MDA), total antioxidant capacity (TAC), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity were determined in the post-thawed samples. In the second experiment, artificial insemination was used to evaluate the fertility and hatchability performances of the post-thawed semen. The results of the first experiment showed that the extenders supplemented with 2, 1 and 0.5 mM of GSH had higher levels (p ≤ 0.05) of motility and motion parameters, PMI, PMF, TAC, CAT and SOD activity and lower abnormal morphology, DNA damage, and lipid peroxidation respectively in comparison to the control group (only extender with semen). Notably, the second experiment showed a higher rate of fertility (p ≤ 0.05) in 2 mM of GSH compared to the control group. It can be concluded that adding 2, 1 and 0.5 mM of glutathione leads to an improvement in the survival of the post-thawed turkey, while 2 mM of GSH can increase the fertility strength of the turkey sperm; hence it can be used to improve fertility and hatchability performance.