Objective To explore and establish a suit of method of cryopreservation of mouse spermatozoa. Methods Two kinds of cryopreservation protection solutions(CPS and RS318) and two thawed methods were studied. Four strains(B 6DF 1,CD-1,DBA, C 57BL/6) of mouse spermatozoa were cooled rapidly and slowly respectively. Then frozen-thawed spermatozoa and oocytes were used for in vitro fertilization(IVF) under the control of fresh sperm. The techniques of culture and embryos transfer were used. Results There was no remarkable difference in the rates of IVF between CPS and RS318 or two thawed methods. The rates of IVF(35%,34%,17%)of tests of B 6DF 1,CD-I,C 57BL/6 mouse spermatozoa cooled rapidly were remarkably lower than those of fresh spermatozoa(60%,59%,34%) and spermatozoa cooled slowly(62%,58%,30%),and the rates of pregnancy in the tests of spermatozoa cooled were remarkably lower than those in fresh spermatozoa except for DBA. Conclusion Mouse spermatozoa cooled slowly is a feasible method, with dry thawed and dilution methods used.