Embryo implantation is a crucial process for successful pregnancy. To date, the mechanism of embryo implantation remains unclear. Ezrin-radixin-moesin-binding protein-50-kDa (EBP50) is a scaffold protein, which has been shown to play an important role in cancer development. Embryo implantation and cancer follow a similar progression. Thus, in this article, we utilized immunohistochemical staining and western blot analyses to examine the spatiotemporal expression and regulation of EBP50 both in the mouse uterus during embryo implantation as well as in other related models. We found that EBP50 was detected in epithelial cells in all of the groups used in our study. During the peri-implantation period, EBP50 mainly localized in apical membranes. At the implantation site (IS) on day 5 (D5) of pregnancy, EBP50 was mainly expressed in the nuclei of stroma cells, whereas from day 6 to day 8 (D6–D8) of pregnancy, the expression of EBP50 was noted in the cytoplasm of decidual cells. The expression of EBP50 was not significantly different in the pseudopregnant uterus and decreased in the uteri subjected to activation of delayed implantation. Artificial decidualization also decreased EBP50 expression. Thus, the expression levels and location were affected by active blastocysts and decidualization during the window of implantation.