Objective: This study evaluated the effects of temperature and storage time on the quality and DNA integrity of freeze-dried sperm from individuals with normozoospermia. Methods: Normal sperm samples from 15 men aged 24 to 40 years were studied. Each sample was divided into six groups: fresh, freezing (frozen in liquid nitrogen), freeze-dried then preserved at room temperature for 1 month (FD-1m-RT), freeze-dried then preserved at room temperature for 2 months (FD-2m-RT), freeze-dried then preserved at 4 °C for 1 month (FD-1m-4 °C), and freeze-dried then preserved at 4 °C for 2 months (FD-2m-4 °C). The morphology, progressive motility, vitality, and DNA integrity of the sperm were evaluated in all groups. Results: In all freeze-dried groups, sperm cells were immotile after rehydration. The freeze-dried groups also showed significantly less sperm vitality than the fresh and frozen groups. Significantly more morphological sperm abnormalities were found in the freeze-dried groups, but freeze-drying did not lead to a significantly higher DNA fragmentation index (DFI). The DFI was significantly higher in the FD-2m-RT group than in the other freeze-dried groups. Conclusion The freeze-drying method preserved the integrity of sperm DNA. The temperature and duration of storage were also identified as factors that influenced the DFI. Accordingly, more research is needed on ways to improve sperm quality in the freeze-drying process.