Rapid freezing and vitrification are becoming popular for human sperm cryopreservation;however, it remains unclear which method is better. The aims of the present study were to determine the optimal trehalose concentration and to compare the cryoprotective effects of rapid freezing and vitrification. The results showed that: 1) The optimal trehalose concentration was 0.25 mol/L;2) The post-thaw recovery rates of total and progressive sperm motilities after rapid freezing (38.6% ± 3.0% and 41.1% ± 5.0%) were significantly higher (P 0.05) than that after vitrification (26.1% ± 3.1% and 27.2% ± 1.3%) when 0.5 mL straws were used;3) However, the recovery rates of total and progressive motilities after rapid freezing in 0.5 mL straw (26.7% ± 9.6% and 26.8% ± 8.7%) were significantly lower (P 0.05) than that after vitrification in a novel straw-in-straw system (43.1% ± 4.2% and 41.8% ± 15.5%);and 4) The post-thaw sperm nuclear DNA damage level after rapid freezing in 0.5 mL straw (8.7% ± 2.8%) was not significantly different from that of sperm after vitrification in the straw-in-straw system (9.2% ± 2.5%). It was concluded that rapid freezing is superior to vitrification when using 0.5 mL straws;however, vitrification is superior to rapid freezing when using the straw-in-straw systems.