【Abstract】Objective Adjust sperm and egg encounter time to improve the fertilization rate of superovulated C57BL/6J. Method (Ⅰ) To deterimine the fertilization rate by routine superovulated method, which as a control group. (Ⅱ) To verify the insemination ability of C57BL/6J male mouse. (Ⅲ) Determination the time of coitus by check vaginal plug at 3 time points after female and male in one cage, to calculate the time for sperm and egg encounter. (Ⅳ)Female C57BL/6J mouse after superovulatory treatment, stayed for 6.5 h before matinge with male mouse. Collected fertilized eggs，calculated the rate of fertilization and compared effects between method changed before and after. Result (Ⅰ) 10.88 fertilized eggs were collected for one mouse in control group; (Ⅱ) Average 20.55 fertilized eggs were collected in the second group, increased 88.87% than control group, that demonstrated the ability of male mouse for getting a high fertilization rate in natural, and the encounter time for sperm and egg was the key factor on fertilization rate; (Ⅲ) For majority mice with plugs at 4 h after cohabitation, the sperm and egg encounter time in control group could be assured that was approximately 7 h; (Ⅳ) Based on the results of above, delayed 6 h for female and male mice feeding together, the mean number of fertilized eggs for one mouse was 22.27, raised 105% than control group. Conclusion Adjusting the sperm and egg encounter time can significantly increase fertilizer ratio of superovulated C57BL/6J mouse.