Background: Freezing is a crucial technique in reproductive science utilized for the preservation of sperm samples. However, the process of freezing and thawing sperm can result in detrimental effects on sperm quality. One of the major mechanisms underlying this decline in sperm quality is the generation of reactive oxygen species during the freeze process. The purpose of the current study was to investigate the effects of magnesium oxide nanopjri_articles on frozen sperm parameters. Methods: Semen samples were collected from 8 fertile men, aged 30 to 42 years, with normozoospermia, following 3 to 5 days of abstinence. The samples were divided into fresh (n=3), freeze (n=3), and control (n=2) groups. Three fresh experimental groups were only exposed to MgO NPs with concentrations of 5, 25, and 50 μg/ml and three freezing experimental groups were frozen after being treated with MgO NPs, thawed, and analyzed after 30 min. Results: Our findings revealed that the progressive movement and vitality of sperm experienced a significant decline, while non-progressive and immotile sperm showed a notable increase in both fresh and frozen experimental groups exposed to MgO NPs. However, the application of MgO NPs during fresh and freezing processes demonstrated an effective preservation of pH, morphology, and DNA fragmentation in sperm cells. Conclusion: The analysis revealed that MgO NPs negatively impact sperm motility and viability in both fresh and freeze analysis. Also, the use of MgO NPs in fresh and frozen processes effectively maintains the pH, morphology, and fragmentation of DNA in sperm cells.