Although equine chorionic gonadotropin (eCG) combined with gonadotropin-releasing hormone (GnRH) is known to improve reproductive rates by increasing the numbers of ovulating oocytes, the actions of these hormones on embryo implantation have not been elucidated. We herein eliminated the effects of embryos by performing embryo transfer and elucidated the effects on embryo implantation after hormonal treatment. The recipient mice were allocated to four groups, including control, eCG, GnRH, and eCG + GnRH groups. Well-developed blastocysts were transferred into recipients, and implantation sites were counted five days after embryo transfer. Our results revealed that embryo implantation rates in the eCG and eCG + GnRH groups were significantly lower than GnRH and control groups; eCG and eCG + GnRH groups didn't differ, and the implantation rate was the lowest in the eCG + GnRH group. When we evaluated estrogen and progesterone concentrations in four groups on embryonic day 0.5 to embryonic day 3.5, we noted that serum estrogen levels in eCG + GnRH group were significantly higher than the other groups, while progesterone levels were significantly lower than the control group, and that the progesterone/estrogen ratio was significantly reduced relative to the control group. Finally, the expression of the endometrial receptivity-related Lif gene was significantly diminished in the eCG + GnRH and eCG groups compared to the other groups. These results suggest that eCG combined with GnRH as a super-ovulatory regimen reduced mouse embryo implantation rates via an estrogen-mediated impairment of endometrial receptivity.