Objective To discuss the effect of in vitro fertilization (IVF) and mouse sperm cryopreservation, to establish a simple and economic frozen system for the genetically engineering mice preservation. Methods Sperm from genetically engineering mice were cryopreserved, IVF was performed using post-thawed sperm, then embryo transfer,to compare the effects of cryopreservation medium、age of male mice and sperm preincubation medium. Results Using CPA as sperm cryopreservation medium, when PM was used thawed-sperm preincubation in IVF, the fertility rates were from 82.49% to 91.43%, when HTF was used thawed-sperm preincubation in IVF, the fertility rates were from14.46% to 27.38%, there was a signification difference between PM and HTF sperm preincubation medium; 10 to 35 weeks male genetically engineering mice sperm were succeed cryopreservation, and positive mice were procreated after 2-cell embryos were transferred; R18S3、CPM and CPA was used to freeze sperm, the fertility rates were 75.85%、88.89% to 94.27%, positive mice were procreated after 2-cell embryos were transferred; 2-cell embryos after IVF were freezed, then thawed and positive mice were procreated after 2-cell embryos were transferred. Conclusion Using CPA as sperm cryopreservation medium, when PM was used thawed-sperm preincubation in IVF, genetically engineering mice sperm were succeed cryopreservation.