The aim of this study is to investigate the methotrexate (MTX) in rat embryonal implantation and its association with Glycodelin A (GdA) and Mucin-1 (MUC-1) expression. For this purpose, 32 pregnant rats were divided into four equal groups: non-pregnant rats in group I (n = 8, control) and pregnant rats in group III (n = 8) were injected intraperitoneal with single dose of normal saline, non-pregnant rats in group II (n = 8) and pregnant rats in group IV (n = 8) were given 0.2 mg i.m. injection of MTX before three months of pregnancy. The dams were killed on 5th day of gestation and uterine horn samples were removed. Following dissection and routine histological preparation, immunohistochemical analysis was carried out. During immunohistochemical examination of the tissue samples prepared from the control and experimental groups, a statistically significant difference was observed between the groups in the luminal-glandular-decidualized epithelium of the uterus with GdA and MUC-1. Finally, in light of our findings, MTX adversely affected the expression of two molecules in Wistar Albino rats embryonal implantation model.