Human semen cryopreservation in the clinical management of male infertility is complicated by cryodamage to spermatozoa. We aimed to clarify the full pattern of cryodamage and evaluate the protective effects of ascorbate and catalase on cryopreserved spermatozoa. Semen samples were collected from 30 fertile males. Each sample was divided into six groups: fresh semen, cryopreserved semen without treatment, and samples cryopreserved with ascorbate (300μM , 600μM), or catalase (200 IU/ml, 400 IU/ml). Spermatozoa were examined for their viability, motility, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), apoptosis (positive for Annexin-V and negative for PI, i.e., Ann+ /PI), and DNA damage (Oliver tail moment, OTM) in the presence or absence of ascorbate or catalase during cryopreservation. In comparison with the fresh spermatozoa, there was a significant decrease in the viability, motility, and MMP, but increase in Ann+/PI-, and OTM in the cryopreserved spermatozoa (P<0.01, or P<0.05). Concurrently, ROS levels in the post-thaw spermatozoa also increased significantly, and this elevation was well correlated with the quality variations of post-thaw spermatozoa (P<0.01, for all). Ascorbate (300 μM) or catalase (200 IU/ml, 400 IU/ml) reduced the ROS levels in post-thaw spermatozoa significantly, compared with those in the control (P<0.05). Furthermore, these antioxidants also prevent those characteristics from being adversely affected (P<0.05). This study demonstrates that the cryopreservation produces various cryodamage to human spermatozoa, possibly through mechanism of ROS. Appropriate ascorbate or catalase supplementation of cryoprotective medium restrains ROS level and the resultant cryodamage.