Objective:To investigate the relationship of the semen separate methods and the clinical outcome of in vitro fertilization and embryo transfer(IVF-ET).Methods:Seventy three cycles of IVF-ET with second infertility were analyzed retrospectively.These cycles were divided into two groups on the basis of semen separate methods:group A(Isolate)42 cycles,group B(Isolate and up swim)31 cycles.Results:There was no significant difference in the the dosage of gonadotrophins(Gn),the days of Gn,the number of oocytes retrieved,the fertilization rate,the cleavage rate and high-quality embryo rate(P>0.05),the embryo implantation rate and pregnancy rate of group A was lower than group B(P<0.05),group A 18.2%(16/88),26.2%(11/42),group B 2.3(20/62),51.6%(16/31).Conclusions:The clinical outcome of in vitro fertilization and embryo transfer with semen separate methods of Isolate and up swim is better than separate methods of Isolate.