The aim of this study was to evaluate the effect of in vitro addition of glutathione in four different concentrations compared to the control group, using two different freezing systems: controlled-rate (AS) and manual (MS) freezer. The parameters evaluated were motility, strength, plasmatic and acrosomal membrane integrity of spermatozoa from twelve stallions. Ejaculates from stallions were collected three times per week, during four weeks. Gel-free semen was diluted in skim milk extender and cooled (16ºC) for 24 h. After cooling, extended semen was centrifuged at 600 x g for 10 min. The supernatant was removed and sperm pellets were re-suspended using the freezing extender with different glutathione concentrations (0, 2.5 mM, 5 mM, 7.5 mM, and 10 mM). Samples were then packed into 0.5 ml straws, which were divided into two parts: one for cryopreservation in a controlled-rate freezer (AS) and the other for a manual system (MS). In this study, the control group showed higher motility and better membrane integrity within treatments for the AS (P < 0.05). Furthermore, when an MS group was evaluated, the 2.5 mM glutathione group demonstrated better preservation for total motility and plasmatic membrane integrity. However, concentrations higher than 2.5 mM were deleterious to the spermatozoa in both controlled and manual freezing systems.