The ability of the neurohormone melatonin to ameliorate cryopreservation-induced damage to spermatozoa has been demonstrated in several domestic species. However, it is unclear how these protective effects are conferred, with improvements in sperm quality ambiguously attributed to the general antioxidant activity of melatonin. To further investigate this phenomenon, ram spermatozoa were diluted in cryomedia with and without melatonin (0 [control], 0.1, 1, 10, and 100 μM) and assessed for motility, viability, DNA integrity, mitochondrial superoxide production, lipid peroxidation, and intracellular reactive oxygen species before freezing and after thawing (0, 3, and 6 h post-thaw). Before freezing, supplementation with melatonin at any concentration had no effect on any measure of sperm quality. However, post-thaw, spermatozoa frozen in the presence of any level of melatonin reduced mitochondrial superoxide production of spermatozoa (P < 0.001), decreased the level of sperm DNA fragmentation (P < 0.001), and increased the percentage of motile spermatozoa (P = 0.035). Melatonin supplementation did not influence the relative levels of lipid peroxidation in the sperm membrane, the levels of intracellular reactive oxygen species, or sperm membrane lipid disorder (P > 0.05). There was no difference in the percentage of viable spermatozoa between treatment groups pre- or post-freeze (P > 0.05). These results suggest that, in the ram, melatonin does not protect the quality of cryopreserved spermatozoa through a nondiscerning scavenging of reactive oxygen species as previously suggested. Rather, melatonin appears to specifically reduce mitochondrial superoxide production, altering sperm functionality, as opposed to merely increasing the percentage of live sperm.