Cryopreservation causes decreased sperm fertility potential due to reactive oxygen species (ROS) production and physical-chemical damage, resulting in reduced sperm viability and motility. The addition of antioxidants to freezing media could protect sperm from cryo-damage, counteracting the harmful effects of ROS. The aim of this study was to assess the effects of curcumin supplementation in freezing medium on preventing cryo-damage in human semen. Semen samples collected from fertile men were cryopreserved in freezing medium supplemented with different concentrations of curcumin (2.5, 5, 10, and 20 μM). After freezing-thawing, sperm parameters, DNA fragmentation, intracellular ROS, and glutathione peroxidase 4 (GPX4) gene expression were evaluated. Supplementation with 20 μM curcumin in freezing medium caused increases in progressive and nonprogressive motility and significant reductions in intracellular ROS and DNA fragmentation in frozen-thawed sperm cells. Following cryopreservation, GPX4 mRNA expression was significantly upregulated in thawed semen supplemented with 20 μM curcumin compared to the control. The results showed that curcumin supplementation in freezing medium was protective against human sperm parameters and sperm DNA, counteracting oxidative damage induced by the freeze-thaw process.