OBJECTIVE: During cryopreservation process, cold shock and cryo-injury affect the fertilizing capacity of the sperm by damaging cell membranes with loss of functional integrity. A longstanding concept for preventing the cryo-damage is to stabilize the plasma membrane by incorporating cholesterol. This study was to determine the effects of cholesterol in freezing media on the motility and functional integrity of human sperm after cryopreservation. METHODS: Control group (non-cholesterol treated) and different concentrations of cholesterol-treated sperm (14 healthy males) were frozen and thawed. After freezing and thawing of sperm, the quality of sperm was evaluated by sperm analysis, acrosome reaction test and sperm chromatin structure assay. RESULTS: When human sperm were incubated in sperm freezing medium (SFM) containing 0.5 microgram cholesterol and then freezing/ thawing, the motility of sperm have significantly improved compared to those untreated cholesterol (33.46+/-1.48% vs. 30.10+/-1.07%, p<0.05). The rate of calcium ionophore-induced acrosome reactions in post-thawed sperm was significantly higher than that (53.60+/-1.60% vs. 47.40+/-1.86%, p<0.05) in SFM containing cholesterol. Sperm chromatin structure assay revealed that DNA damage to the sperm in the cholesterol-treated group was lower than that of non-treated group. CONCLUSION: These results suggest that increased cholesterol content of sperm plasma membrane by supplementation of cholesterol in SFM improves sperm motility, capacitation status, and DNA integrity. Therefore, addition of cholesterol into SFM could be a useful for protecting human sperm from cold shock and cryo-injury during cryopreservation.