Oxidative stress imbalance potentially leads to damage of the structure of the cell and macromolecules such as plasma membrane components, proteins, and DNA. The plasma membrane of the sperm cell, which has high levels of polyunsaturated fatty acids, renders it particularly sensitive to free radical–mediated attacks. The freezing and subsequent thawing of sperm is a physically stressful process carried out during routine procedures in assisted reproduction techniques, which results in a highly variable and unpredictable reduction in the number of motile sperm cells. Subsequently, oxidative status can positively or negatively affect the motility, viability, and fertilizing capacity of thawed sperm. These effects are counteracted by various oxidative defense enzymes and anti-oxidants such as glutathione peroxidase isoforms GPx1 and GPx4, glutathione reductase (GR), and cellular glutathione (reduced) (GSH). In this way, oxidative status could represent a predictive marker of sperm quality following the freeze-thaw process. This study was based on 56 human sperm samples. We observed direct positive and negative relationships between the postthaw motile sperm recovery rate and GPx1 and GPx4 expression and activity, on the one hand, and GSH concentrations, on the other. No correlation was found between this recovery rate and GR or basic semen parameters. Predictive values clearly demonstrate that, among the molecules analyzed, the most accurate diagnoses result when analyses are conducted for GPx1 and GPx1 messenger RNA expression, GPx1 and GPx4 enzymatic activity, and GSH concentration. In conclusion, a reserve of glutathione, together with GPx expression, is necessary to eliminate free radicals using GSH or a like structural protein and seems to be essential for a good postthaw recovery. These molecules can be employed as indicators of postthaw sperm quality.