This study evaluated the effects of supplementing 5.0 mM glutathione (GSH) to the media during semen thawing and culture on forward progressive spermatozoa motility, viability, and DNA fragmentation. Pellets of frozen boar semen were thawed using a PBS wash procedure at 39°C. Spermatozoa were analyzed for forward progressive motility, viability, and DNA fragmentation by the Comet assay at 0.5 h and 6.0 h post-thawing. Supplementation of 5.0 mM GSH had no significant effect on forward progressive motility, viability, or DNA fragmentation at 0.5 h post-thawing compared to the control. At 6.0 h after thawing there was no significant difference in viability between the control and the 5.0 mM GSH supplemented group. Forward progressive spermatozoa motility was significantly less (P < 0.05) at 6.0 h after thawing in the 5.0 mM GSH supplemented group compared to the control. DNA fragmentation was significantly higher (P < 0.05) at 6 h after thawing in the 5.0 mM GSH supplemented group compared to the control. These results indicate that supplementing 5.0 mM of GSH to the thawing and culture media significantly decreases (P < 0.05) spermatozoa forward progressive motility and significantly increases (P < 0.05) DNA fragmentation