Agrin is the heparan sulfate proteoglycan (HSPG) that is well known as the molecule that aggregates acetylcholine receptor (AChR) through muscle specific kinase (MuSK) and rapsyn at neuromuscular junctions. HSPGs are spatiotemporally expressed in embryonic and maternal tissues during implantation. The present study examined the role of agrin in the mouse embryo using leukemia inhibitory factor (LIF)-deficient mice, which show complete sterility. Agrin was detected widely in the cytoplasm of uterine luminal epithelial cells at the third day of pregnancy (Day 3) and Day 4. At Day 5, agrin moved to the apical surface of the luminal epithelium. This migration was not found in LIF-deficient mice. AChR was also found in the apical surface of the uterine epithelium at Day 4 and Day 5 in normal mice. LIF-deficient mice did not show this pattern of expression. Only nAChR b1 subunit mRNA was increased at Day 5 in normal mice. Furthermore, acetylcholinesterase was active in the uterine stroma of normal mice throughout the implantation period and was exclusively active in the uterine epithelium at Day 4. Taken together, agrin signaling was activated in the uterus during embryo implantation in the mice. Here, we suggest that the agrin pathway is involved in closure of the uterine epithelium toward placentation.