Abstract: In this paper, DMSO was used as cryoprotectant for cryopreservation of Sparus macrocephalus spermatozoa in 0.5 mL straws. Detection of DNA damage in response to a cryopreservation process in Sparus macrocephalus spermatozoa was also carried out. The results demonstrated that there were no significant differences between frozen-thawed sperm conserved by Cortland solution diluted with 5%, 10%, 15%, 20% DMSO and fresh sperm in motility. The best motility of frozen-thawed sperm were obtained when DMSO concentration was 10%, and the activation rate, moving time, living time and fertilization rate of frozen-thawed sperm were 92.91±1.25%, 39.90±2.70 min, 53.82±2.84 min and 89.35±1.99% respectively. However, a significant drop in sperm motility and fertilization rate was observed in sperm cryopreserved with 25% and 30% DMSO. The comet rate and damage coefficient of frozen-thawed sperm conserved with 5%, 10%, 15%, 20% DMSO was similar to fresh sperm, but at 25% and 30% were significantly differed to fresh sperm. In fact, there was a positive correlation between comet rate of frozen-thawed sperm and concentration of DMSO in protocol. The majority of sperm with DNA damage within the nucleus were slightly and mildly damaged, while minorities were heavily damaged. Few were totally damaged, and only occurred under the conditions of 25% and 30% DMSO. Our analysis suggests that high concentration of DMSO is the main factor that causes the DNA damage in frozen-thawed sperm nucleus.