Glutathione (GSH) and melatonin (MLT) are two antioxidants that have been described as having a protective role against oxidative stress in several animal species, improving sperm quality and survival after cryopreservation. Since there is no knowledge on the simultaneous use of these two substances concerning its effects on ram semen cryopreservation, this study aimed to evaluate the effects of MLT, GSH, or their association, on in vitro parameters of ram semen after thawing. During eight weekly sessions, the semen from three Merino rams was pooled, divided into four aliquots, and diluted with an egg yolk-based semen extender without (control) and with antioxidants (MLT 1mM, GSH 5mM, and MLT 1mM + GSH 5mM). Diluted semen was evaluated before and after freezing process. Supplementation of fresh semen with GSH at 5 mM had a deleterious effect on total motility progressive (TPM), curvilinear velocity (VCL), straight-line velocity (VSL), average-path velocity (VAP), linearity (LIN), straightness (STR) and increased slow spermatozoon (%). MLT at 1 mM also has a negative effect on TPM, VSL, and STR in fresh semen. In thawed semen, 1 mM of melatonin increased the TPM regarding the group control. Also, the combination of both antioxidants attenuates the negative effect of GSH 5mM on TPM, VSL and BCF. These results indicate that 5mM GSH impairs or does not improve sperm kinetic parameters in fresh semen and suggest that melatonin when combined with GSH plays a protective role on these effects.