Objective To explore whether superovulation impairs the process of pregnancy establishment in mice by changing the intrauterine environment.   Methods The implantation and pregnancy of superovulated and normal mice were compared. The superovulated mice were subjected to unilateral tubal ligation on day 0.5 and blastocysts were transplanted to the other uterine horn on day 2.5. The number of implantation sites of bilateral uterine horn was compared. The differences between preimplantation uteri of superovulated and normal pseudopregnancy mice were compared by tissue sections and high-throughput sequencing.Bioinformatics analysis was performed on the differentially expressed genes in two groups.   Results Compared with the control group, the pregnancy rate of mice in the superovulation group decreased significantly. The number of implantation sites in the superovulation group was higher than the control. There was no significant difference in the pregnancy rate of the uterine horn between the control side and the transplanted side of the superovulated mice. The endometrium was thinned and the number of glands was reduced in superovulated pseudopregnancy mice. The gene expression patterns of preimplantation uterus in superovulation pseudopregnancy and normal pseudopregnancy mice were different. There were 1097 significantly differentially expressed genes, including 752 up-regulated genes and 345 down-regulated genes. Bioinformatics analysis showed that differentially expressed genes are mainly involved in biological processes, such as decidualization, response to progesterone, positive regulation of angiogenesis. They were mainly enriched in FoxO signaling pathway, cell cycle pathway and steroid   biosynthesis pathway.   Conclusion Superovulation impaired the process of establishing pregnancy and altered the gene expression patterns of biomarker of uterine receptivity in mice.