Objective The experiments were carried out to develop an improved in vitro fertilization (IVF) system for Kunmimg mice by comparing the effect of fertilization media and treatment methods on IVF of mature oocytes and the effect of three culture media for early embryo development. Methods Sperm was prepared by sqeezing cauda epididymidis from male mouse and mixed with mature oocyte from superovulated female mouse in T6 or FM media, and mature oocytes were treated with hyaluronidase for removing cumulus cells. The control was not treated before fertilization. The IVF embryos were cultured in M16, modified CZB media (mCZB) or modified M16 media (mM16) media. Results The fertilization rate in FM media was significantly higher than that in T6 media (92 3% vs. 64 7%). The fertilization rate of treatment method to mature oocyte without hyaluronidase before fertilization was higher than that of hyaluronidase treatment method (64 7% vs. 59 2%), but no significant difference was found. The developmental rate of morulae and blastocysts in M16 media (55 3% and 35 6%) were significantly higher than that in mCZB media (17 3% and 14 7%). Conclusion It is indicated that FM medium is more appropriate for in vitro fertilization of oocytes, and it is not necessary to remove the cumulus cells of mature oocyte by hyaluronidase. MM16 media is the most appro priate for in vitro culture of early embryos of Kunming mice, and hypotaurine can efficiently overcome embryo development retardation after 2 cell stage. reofearlyembryos