<p>Background: PON3 is associated with high-density lipoprotein (HDL) and plays a protective role against oxidative damage to low-density lipoprotein (LDL). The purpose of the current study was to investigate the effect of semen paraoxonase-3 (PON3) concentration on sperm parameters and DNA fragmentation in both fresh or post-thaw semen.&nbsp;<br /> Methods: This study analyzed PON3 levels in 30 normospermic semen samples obtained from men who attended Dezful Infertility Center and provided informed consent. The samples were analyzed before and after cryopreservation for sperm motility, DNA fragmentation, and seminal PON3 levels. Data analysis was performed using SPSS software and statistical differences were evaluated using ANOVA. A p&le;0.05 was considered statistically significant.&nbsp;<br /> Results: There was no significant difference in sperm morphology (p=0.37) and count (p=0.25) before and after freezing at different levels of PON3. The highest levels of progressive motility were observed in samples with the highest PON3 concentrations both before (p=0.01) and after freezing (p=0.02), whereas non-progressive motility was significantly greater in samples with the lowest PON3 concentrations at both time points (p=0.01). Sperm DNA fragmentation significantly decreased before or after freezing as PON3 levels increased (p=0.03).<br /> Conclusion: This study suggests that PON3 levels may serve as an indicator for both sperm motility and DNA fragmentation pre- or post-cryopreservation, potentially contributing to future clinical research.</p>