This study explores the regulatory role of proline-rich acidic protein 1 (Prap1) effects on embryo implantation. A high estrogen model and ovariectomized mice were employed to demonstrate that estrogen regulates Prap1 expression. Uterine tissues were collected from E 1.5 (the presence of a vaginal plug was recorded as embryonal day 0.5, E 0.5) to E 7.5 to detect the Prap1 expression pattern in early pregnancy using qRT-PCR. Embryo adhesion was assessed through uterine perfusion of PRAP1 protein and Prap1 overexpression in endometrial epithelial cells (EECs). The data showed that Prap1 expression was increased in the uterus with high estrogen levels. Prap1 expression was specifically reduced during early implantation. Overexpression of Prap1 in EECs also reduced the embryo adhesions. The differentially expressed genes obtained by RNA-seq were enriched in extracellular matrix and cell adhesion. Muc1 expression was increased in EECs overexpressing Prap1 by RNA-seq and qRT-PCR. Similarly, O-glycosylation biosynthesis was enriched, and glycosylation-related genes were upregulated. Our results demonstrate that Prap1 was regulated by estrogen and an increase in PRAP1 before implantation affected embryo adhesion by regulating the expression of Muc1 and extracellular matrix-related genes, leading to embryo implantation failure. Our results provide a new insight into estrogen regulation of embryo implantation.