The effects of temperatures, durations of treatment, and derivations from spermatophores or spermaries on in vitro acrosome reaction of the spermatozoa in the Chinese mitten crab Eriocheir sinensis were investigated. The results showed that the different temperatures resulted in extremely significant differences (p < 0.01) in the time of beginning acrosome reaction, the time of the maximum percentage of acrosome reaction, and the maximum percentage of acrosome reaction of the spermatozoa from spermatophores; and the low temperature (-20, -80 degrees C and liquid nitrogen) induced acrosome reaction of more than 90% spermatozoa while 15 and 4 degrees C didn't. Similar results occur in the spermatozoa, treated with -80 degrees C for 15 min, from spermaries but the time of beginning acrosome reaction and the time of the maximum percentage of acrosome reaction were obviously longer than those from spermatophores. In conclusion, low temperature can induce acrosome reaction, which is a novel and efficient operating method of inducing acrosome reaction; the spermatozoa might be affected physiologically to capacitate with chilling. The study may be beneficial to new understandings of mechanism of acrosome reaction and provide the foundational material for artificial fertilization and breeding of this crab and other commercial aquatic crustaceans.